Synthetic binding pairs comprising cucurbituril derivatives and polyammonium compounds and uses thereof

ABSTRACT

Derivatized cucurbiturils and cucurbituril assemblies formed thereby are disclosed. Also disclosed are binding pairs of the disclosed cucurbituril assemblies and polyamine structures, which are highly advantageous over the presently known affinity pairs and therefore can be efficiently utilized in a myriad of applications.

RELATED APPLICATIONS

This application is a National Phase Application of PCT Application No.PCT/IL2004/000796 having International Filing Date of Sep. 5, 2004,which claims priority from U.S. Provisional Patent Application No.60/499,735, filed on Sep. 4, 2003, and U.S. Provisional PatentApplication No. 60/535,829, filed on Jan. 13, 2004. The contents of theabove Applications are all incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to novel synthetic binding pairs, tomethods of preparing same and to uses thereof in various applicationssuch as, but not limited to, isolation and purification of biologicalmolecules via affinity chromatography, immunohistochemical staining,introducing multiple labels into tissues, localizing hormone bindingsites, flow cytometry, in situ localization and hybridization, radio-,enzyme-, and fluorescent immunoassays, neuronal tracing, geneticmapping, hybridoma screening, purification of cell surface antigens,coupling of antibodies and antigens to solid supports, examination ofmembrane vesicle orientation, and drug delivery.

High-affinity and specificity pairs are of great importance in bothresearch and industrial endeavors in fields such as chemistry and, inparticular, in the biological and medical sciences. Affinitychromatography alone is a valuable tool for separating and purifyingbiological materials from solution. Affinity chromatography techniquetypically involves an affinity pair, of which one component (oftentimesreferred to as the ligand) is immobilized by attaching it to aninsoluble support. The other component, when passed through a columnwithin a mixture of components in solution, is selectively absorbed tothe attached component by forming a complex therewith and is thusisolated from the solution. The second component may subsequently beeluted from the solid support by a number of procedures resulting in thedissociation of the affinity pair.

This technique is widely used to isolate biomolecules such as peptides,proteins, enzymes, inhibitors, antibodies, antigens, hormones,carbohydrates and many more, based on specific interactions formedbetween affinity pairs of such biomolecules. Known examples are thehigh-affinity and specificity interactions of antibody-hapten pairs, andin particular, of the avidin-biotin (Av-B) pair (Wilchek M, MethodsEnzymol., entire Volume 184, 1990, incorporated by reference as if fullyset forth herein).

The Av-B high affinity complexation and the consequent stability of itsnon-covalent interactions (K_(D) of 10¹⁵ M⁻¹) has become the basis of abroad variety of bioanalytical applications and a common tool in almostany molecular biology laboratory. The main applications where Av-Bcomplexes have been used extensively include, for example, isolation viaaffinity chromatography, localization via cytochemistry, cell cytometry,in situ hybridization and blotting technologies, diagnostics viaimmunoassay, histochemistry and histopathology, and gene probes. Inaddition, the Av-B complexes have been also applied in the hybridomatechnology, in the design of bioaffinity sensors, in affinity targeting,drug delivery, cross linking, immobilization, fusogenic studies,screening of combinatorial libraries, in vivo tissue imaging and manymore.

Biotin is a relatively small molecule, a member of the Vitamin B family(formerly known as Vitamin H), whereby avidin is a ubiquitous 66 kDtetrameric protein found in egg whites. A key principle of the Av-Btechnology is that both avidin and biotin can be chemically linked to avariety of either small or large molecules without disrupting thebinding constant therebetween. For example, many reporter groups havebeen covalently attached to avidin, including fluorescent groups,electron-dense markers, enzymes, various binding protein, and varioussolid supports including magnetic beads. Likewise, since the carboxylgroup of biotin is not essential for binding, it has been used eitherdirectly or through a spacer fragment, to synthesize many compoundsincluding proteins, DNA and RNA molecules, with a covalently attachedbiotin moiety.

By covalently attaching a biotin molecule, a reaction known asbiotinylation, one can “tag” an otherwise untraceable molecule or abiochemical entity and turn it into a probe that can be recognized by alabeled detection reagent or an affinity-capture matrix. Once taggedwith biotin, a molecule of interest, such as a peptide, a protein, anantibody, a drug, a polynucleotide, a polysaccharide or another receptorligands, can be used to probe complex systems and mixtures, cells andtissues, as well as protein and nucleic acid blots and arrays. Thistagged molecule can then be detected with the appropriate avidinconjugate that has been labeled with a chromophore/fluorophore, enzymeor other solid and/or magnetic matrices and particles. Biotinylatedmolecules can also be captured with various forms of immobilized avidinor streptavidin, and other modified forms of avidin.

Although binding of biotin to native avidin or streptavidin isessentially irreversible, appropriately modified avidins can bindbiotinylated probes reversibly, making them valuable reagents forisolating and purifying biotinylated molecules from complexed mixtures(Morag E, Bayer E A, Wilchek M. Biochem J 316, 193-199 (1996)).

Many strategies are available for applying the Av-B technology in agiven experimental system. Representative examples of these strategiesare presented in FIG. 1. Thus, in one exemplary strategy (FIG. 1,Strategy A) avidin is attached to a probe, either directly, by covalentbonding, or indirectly, via interaction with a biotinylated probe, andthe target molecule is directly bound to biotin. The biotinylated targetmolecule forms a complex with the avidin probe and is thus analyzed. Inanother exemplary strategy (FIG. 1, Strategy B) a target molecule isattached to a ligand which is covalently bound to either biotin oravidin in order to generate a noncovalent linkage to an avidin-probeconjugate, as described hereinabove, or a biotin probe conjugate,respectively. In yet another exemplary strategy (FIG. 1, Strategy C) thesame principles as in B are utilized, but the target molecule is furtherattached to a binder that is specific to the first ligand, thusgenerating a longer chain of interactions.

However, although technologies utilizing the Av-B affinity pair has beenextensively used over the past two decades, they suffer severaldisadvantages, the following lists a few.

The Av-B binding couple exhibits a disadvantageous high molecularweight, with 58-76 kD for Avidin and 244 D for Biotin. Such a highmolecular weight may lead to loss of resolution in separationtechniques, analytical gels and other applications where the studied andcompared components are small relative to avidin.

The fact that avidin and streptavidin are large biologicalmacromolecules, characterized by a complexed yet inflexible structure,renders these biomolecules susceptible to interactions with many othersmall molecules and biomolecules. For example, due to the molecularorientation of the binding sites, less than four molecules of biotinactually bind to one avidin molecule. The few binding sites on avidinare also the sites where chemical modification takes place, limiting thecapacity for labels and affecting immobilization properties. Avidin mayalso bind many other biomolecules non-specifically. This is especiallysignificant in the case of preparation of oligonucleotide microarrays inwhich non-biotin modified oligonucleotides bind non-specifically toavidin, leading to spurious results.

Furthermore, the fact that proteins are sensitive to chemical andphysical conditions renders the use of avidin or streptavidin limited totechnologies that involve mild conditions and limits the use of the Av-Baffinity pair to aqueous systems at close to physiological temperature.

In addition, Av-B systems suffer from lack of transparency in the UVregion and background fluorescence, and therefore cannot be used inexperiments where detection is depending on various light measurements.

The conjugation chemistry of avidin is rather limited to reactions thatare compatible with polypeptides, and thus limits the chemistry by whichavidin can be attached to various molecules and materials.

The resulting affinity of the Av-B system has an inflexible bindingconstant of K_(D)=10⁻¹⁵ M, which limits their use to applications thatrequire strong binding without the possibility for fine-tuning.

The high binding affinity decreases rapidly (100-1000 fold) when abiomolecule is coupled to biotin.

Finally, the one-step binding of the Av-B couple is practicallyirreversible, unless large quantities of free biotin are applied. Theirreversible nature of the binding limits the ways by which thedissociation of the conjugate can be achieved.

Cucurbiturils are macrocyclic cavitand compounds that are typicallyformed by reacting a number of glycoluril units and formaldehyde unitsunder acidic conditions. For example, Cucurbit[6]uril, also known asCB[6] (FIG. 2, Compound 1), is typically prepared by reacting sixglycoluril molecules, (FIG. 2, Compound 2) and twelve formaldehydeunits, in the presence of a concentrated acid, as is illustrated in FIG.2.

Cucurbiturils (CBs) in general are known since 1905 (Behrend et al.,Liebigs Ann. Chem. 1905, 339, 1), and were first characterized by Mockand co-workers in 1981 (Mocket et al., J. Am. Chem. Soc. 1981, 103,7367). Several substituted cucurbiturils and homologues, collectivelyreferred to as CB[n] whereby n represents the number of glycoluril unitsin the CB and typically ranges from 5 to 8, have also been prepared andcharacterized (Kim, et al., J. Am. Chem. Soc. 2000, 122, 540).

Cucurbiturils, either substituted or unsubstituted, are typicallycharacterized by a hydrophobic cavity that is accessible through twoidentical, polar, carbonyl-fringed portals. This feature, when coupledwith the high yield synthesis of, for example, CB[6] (82%), suggestedthat the formation of CB[6] is governed by a thermodynamic preferencefor CB[6] (Buschmann et al., German Patent DE 196 03 377 A1, 1997).Other studies further indicated that the ring order and by-productpopulation proportions in cucurbiturils syntheses are determined by theglycoluril and aldehyde building-blocks substitution (see, U.S. Pat. No.6,639,069, and Chakraborty et al., J. Am. Chem. Soc. 2002, 124, pp.8297-8306).

Although cucurbiturils are easily prepared via an acid-catalyzedcondensation of the appropriate glycolurils with formaldehyde, thesemacropolycyclic compounds are typically obtained in the form of complexmixtures that further contain many cyclic and acyclic oligomers andpolymers, including insoluble polymers.

The presently known methods of isolating CB[n]s from their reactionmixtures are based mainly on differential solubility in various solventsand on fractional crystallization, methods which suffer from lowefficiency in terms of cost and yield. These methods, however, areoftentimes not suitable for isolation and purification of substitutedCB[n]s. The use of alternative purification or isolation methods suchas, for example, column chromatography is, in most cases, inefficientand difficult to practice due to the high polarity and limitedsolubility of these compounds.

Thus, the isolation of pure CB[n]s has become the major impediment totheir availability, particularly when large-scale synthesis is required.

U.S. Pat. No. 6,365,734 describes the preparation and separation ofvarious CB[n] homologues and derivatives. These methods involvemanipulation of reaction conditions, e.g. acidity and temperature, whichcause a shift in the proportions between various major and minorproducts, yet these manipulations do not provide an efficient method forobtaining, in substantial amounts, minor, thermodynamicallydisadvantageous CB[n]s, which are typically formed in traces amounts.

The rigid structure and the combination of a hydrophobic cavity withpolar portals allow the cucurbiturils to act as cavitands hostingvarious molecules and cations, and thus render the CB[n]s attractivesynthetic receptors and useful building blocks of various supramolecularstructures.

Due to the intricate recognition characteristics of CB[n], many studieswere aimed at synthesizing homologues and derivatives of CB[n] withvarying ring order and substituents. Nevertheless, the domination of onemajor product, and the practical difficulty in separating the moredesired yet minor products, presented major restrictions on the path toobtaining rare CB[n]s.

As mentioned hereinabove, CB[n]s are characterized by two “oculi”,having a 400 pm diameter in the case of CB[6]. These openings allow theentrance of small molecules into the cavity and thus enable an affinitybinding of these molecules to the cavitand. Although simple aliphaticcompounds can thus be bound, the most strong and efficient affinitybinding in the cavity of CB[n]s has been observed with alkylammoniumions (Mock and Shih, J. Org. Chem. 1983, 48, p. 3618).

The exceptional binding affinity between alkylammonium ions and CB[n]shas been attributed to the ion-dipole interaction between the ammoniummoiety and the oculi carbonyls, and to the hydrophobic interactionsformed when the alkyl moiety displaces solvent molecules from within thecavity (Mock and Shih, J. Org. Chem. 1986, 51, p. 4440).

The combination of complexation and recognition interactions lendsitself to a range of strong and highly specific entrapping abilities ofn-alkylammonium ions by various cucurbiturils. The symmetric structureof the two “oculi” further offers recognition factors, as evident fromthe interaction of n-alkyldiammonium ions with CB[n] (Mock, W. L. inComprehensive Supramolecular Chemistry; Vögtle, F., Ed.; Elsevier Press:New York, 1996; Vol. 2, pp 477-493).

Studies have shown (Mock and Shih, J. Org. Chem. 1986, 51, p. 4440) thatthe binding strength between alkylammonium ions and cucurbiturilsdepends on the chain length of the alkyl group of n-alkylammonium andn-alkyldiammonium ions, whereby the optimal chain length was found to be4, for n-alkylammonium, and 5-6 for n-alkyldiammonium, with the latterpossessing ten-fold higher affinity to CB[6] as compared with that ofthe first.

The binding affinity between alkylammonium ions and CB[n]s was found tobe further affected by stearic hindrance and ring size, in cases ofsubstituted and cyclic ammonium ions (Mock and Shih, J. Org. Chem. 1986,51, p. 4440). Thus, the presence of two or more amine groups in thealkyl chain, was found to affect the binding rate and dynamics, being adomain of distinguished states sensitive to chemical (e.g., pH) andphysical (e.g., temperature) conditions, and thus renderingCB[n]-polyamine systems highly suitable for molecular switches andquantum binding.

CB[n]s and protonated polyaminoalkanes form stable host-guest complexes,exhibiting sub-micromolar affinity dissociation constants (K_(D)) in therange of e.g., 10⁻⁵-10⁻⁷ M (Mock et al, J. Org. Chem. 1986, 51, 4440)for protonated diaminoalkanes, such as 1,4-diaminobutane,1,5-diaminopentane and 1,6-diaminohexane. This property has beenextensively used by Kim [1] and others [2] to construct manysupramolecular assemblies, including catenanes, rotaxanes, andpseudopolyrotaxanes.

Nevertheless, although the high affinity between CB[n]s and polyamineshas been studied, the use of CB[n]s-polyamines affinity pairs both inbasic research and in biotechnology and medicine applications such as,for example, tagging and labeling, purification, cytometry, drugdelivery, administration and other applications, has never beensuggested nor practiced hitherto.

While conceiving the present invention, it was envisioned that the highaffinity between CB[n]s and polyamines, the versatile and controllablecharacteristics of CB[n]s and polyamines and the effect of thesecharacteristics on the affinity could be beneficially used in a myriadapplication, while circumventing the limitations associated with thepresently used Av-B technology.

There is thus a widely recognized need for, and it would be highlyadvantageous to have a practical, fast, general and cost effectivemethod for separation and purification of CB[n] homologues orderivatives, which would enable to obtain CB[n]s and in particular rareand thermodynamically disadvantageous forms of CB[n]s, in substantialyields, while circumventing the above limitations, and which wouldenable to efficiently use such CB[n]s to form high-affinity pairs ofCB[n] entities and protonated polyaminoalkanes, devoid of thelimitations associated with the presently known affinity pairs.

SUMMARY OF THE INVENTION

While reducing the present invention to practice, it was uncovered thatderivatized cucurbiturils can be efficiently prepared and utilized forforming cucurbituril assemblies. These cucurbituril assemblies, in turn,can form affinity binding pairs with polyamines structures that aredesigned capable of binding to these assemblies. The resulting affinitypairs are highly advantageous over the presently known affinity pairsand therefore can be efficiently utilized in a myriad of application.

Thus, according to one aspect of the present invention there is provideda cucurbituril comprising at least one functional group covalentlyattached thereto, whereby the at least one functional group being forforming an assembly of at least two cucurbiturils.

According to further features in preferred embodiments of the inventiondescribed below, the cucurbituril is selected from the group consistingof CB[5], CB[6], CB[7], CB[8] and CB[n], wherein n is an integer thatequals to or is lower than 20. Preferably the cucurbituril is selectedfrom the group consisting of CB[5], CB[6], CB[7] and CB[8].

According to still further features in the described preferredembodiments the assembly is selected from the group consisting of adimer, a trimer, a polymer, an oligomer, a dendrimer and a cluster ofthe cucurbituril.

According to still further features in the described preferredembodiments the at least one functional group is selected from the groupconsisting of amine, halide, sulfonate, sulfinyl, phosphonate,phosphate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,thioaryloxy, cyano, nitro, azo, sulfonamide, carboxylate, thiocarbamate,urea, thiourea, carbamate, C-amide, N-amide, guanyl, guanidine andhydrazine.

According to still further features in the described preferredembodiments the at least one functional group is amine, preferably asecondary amine.

According to still further features in the described preferredembodiments the at least one functional group is attached to thecucurbituril via a spacer.

The spacer can be, for example, an alkyl having 1 to 20 carbon atoms, analkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to20 carbon atoms, an alkoxy having 1 to 20 carbon atoms, an aminoalkylhaving 1 to 20 carbon atoms, a cycloalkyl having 5 to 20 atoms, aheteroalicyclic having 4 to 20 carbon atoms, an aryl having 6 to 20carbon atoms and/or a heteroaryl having 6 to 20 carbon atoms.

According to still further features in the described preferredembodiments the spacer is a heteroalicyclic having a secondary amine asa functional group that is incorporated therein.

According to still further features in the described preferredembodiments the spacer comprises a pyrrolidine ring being fused to thecucurbituril.

According to still further features in the described preferredembodiments the cucurbituril assembly comprises an assembling unit thatis covalently attached to each of the at least two cucurbiturils viaeach of the at least one functional group.

According to still further features in the described preferredembodiments the assembling unit comprises at least one subunit selectedfrom the group consisting of cycloalkyl, heteroalicyclic, aryl,heteroaryl, polyaryl, polyheteroaryl, amide, sulfonamide, phosphonate,phosphate, carboxyl, thiocarboxyl, carbamyl, thiocarbamyl, ureido,thioureido, and hydrazine.

According to still further features in the described preferredembodiments the assembling unit comprises at least one triazine.

The derivatized cucurbiturils described above can be presented,according to another aspect of the present invention, by the generalformula:

wherein:

n is an integer from 5 to 20;

each of X₁, X₂ . . . X_(n) and E₁, E₂ . . . E_(n) is independentlyselected from the group of O, S and NR′; and

each of R′, A₁, A₂ . . . A_(n), D₁, D₂ . . . D_(n), Y₁, Y₂ . . . Y_(n)and Z₁, Z₂ . . . Z_(n) is independently selected from the groupconsisting of a hydrogen, an alkyl having 1 to 20 carbon atoms, analkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to20 carbon atoms, an alkoxy having 1 to 20 carbon atoms, an aminoalkylhaving 1 to 20 carbon atoms, a cycloalkyl having 5 to 20 atoms, aheteroalicyclic having 4 to 20 carbon atoms, an aryl having 6 to 20carbon atoms and a heteroaryl having 6 to 20 carbon atoms,

whereas at least one of the A₁, A₂ . . . A_(n) and D₁, D₂ . . . D_(n)comprises a functional group as described hereinabove, the functionalgroup being for forming an assembly comprising at least two derivatizedcucurbiturils having the general formula above.

Preferably, n is an integer from 5 to 8.

Further preferably, each of the X₁, X₂, . . . X_(n), E₁, E₂ . . . andE_(n) is O.

Further preferably, either each of the Y₁, Y₂ . . . Y_(n) or each of theZ₁, Z₂, . . . Z_(n) is hydrogen.

Further preferably, each of the Y₁, Y₂ . . . Y_(n) and the Z₁, Z₂ . . .Z_(n) is hydrogen.

According to another aspect of the present invention there is provided acucurbituril assembly comprising at least two cucurbiturils and at leastone assembling unit, as described above, being covalently attached toeach of the at least two cucurbiturils.

According to further features in preferred embodiments of the inventiondescribed below, each of the at least two cucurbiturils comprises atleast one functional group, as described above, and the at least oneassembling unit is covalently attached to each of the at least twocucurbiturils via the at least one functional group.

The cucurbituril assembly can be selected from the group consisting of adimer, a trimer, a polymer, an oligomer, a dendrimer and a cluster ofthe at least two cucurbiturils.

The at least one assembling unit comprises, for example, at least onesubunit selected from the group consisting of cycloalkyl,heteroalicyclic, aryl, heteroaryl, polyaryl, polyheteroaryl, amide,sulfonamide, phosphonate, phosphate, carboxyl, thiocarboxyl, carbamyl,thiocarbamyl, ureido, thioureido, and hydrazine.

The cucurbituril assembly may further comprise at least one functionalmoiety, as is detailed hereinbelow, being attached thereto.

The at least one functional moiety can be attached to at least one ofthe at least two cucurbiturils or to an assembling unit.

According to still another aspect of the present invention there isprovided a polyamine structure being capable of binding to at least onecucurbituril, as described hereinabove, which comprises at least twoamine groups and at least one threading moiety terminating and/orinterrupted by the at least two amine groups, the at least one threadingmoiety being suitably sized to the at least one cucurbituril.

Each of the threading moieties can be, for example, an alkyl having 1 to20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, analkynyl group having 2 to 20 carbon atoms, an alkoxy having 1 to 20carbon atoms, an aminoalkyl having 1 to 20 carbon atoms, a cycloalkylhaving 4 to 20 atoms, a heteroalicyclic having 4 to 7 carbon atoms, anaryl having 6 to 20 carbon atoms or a heteroaryl having 5 to 20 carbonatoms.

Preferably the threading moieties comprise an alkyl having 1 to 20carbon atoms, more preferably an alkyl having 1 to 10 carbon atoms, and,more preferably, an alkyl having 5 to 6 carbon atoms.

Further preferably, the threading moieties have a rigid structure andthus comprise, for example, at least one alkynyl having 2 to 6 carbonatoms.

According to further features in preferred embodiments of the inventiondescribed below, the polyamine structure comprises at least twothreading moieties terminating and/or interrupted by the at least twoamino group, which are covalently attached therebetween via a branchingunit.

The branching unit can be, for example, an amine, a branched alkyl, acycloalkyl, a heteroalicyclic, a branched alkenyl, an aryl, aheteroaryl, a silyl, a silicate, a boryl, a borate, a carbamate, athiocarbamate, a C-amide, a N-amide, a S-sulfonamide, an N-sulfonamide,urea, hydrazine, guanyl and/or guanidine.

The polyamine structure can further comprise at least one functionalmoiety attached thereto, as is detailed hereinbelow.

The functional moiety can be attached to at least one of the threadingmoieties and/or to the branching unit.

The polyamine structure of the present invention is further capable ofbinding to at least two cucurbiturils, which form a cucurbiturilassembly, as described above.

Such a polyamine structure preferably comprises at least two threadingmoieties terminating and/or interrupted by the at least two amino group,wherein each of the at least two threading moieties is capable ofbinding to each of the at least two cucurbiturils in the assembly.Preferably, the at least two threading units are covalently attachedtherebetween via a branching unit, as described above, whereby thebranching unit is suitably sized to the cucurbituril assembly.

According to yet another aspect of the present invention there isprovided an affinity pair, which comprises the cucurbituril assembly andthe polyamine structure of the present invention, as described above.

The affinity pair can further comprise one or more functional moietiesattached thereto, ether via the cucurbituril assembly, the assemblingunit, the threading moiety and/or the branching moiety.

The affinity pair of the present invention preferably has a dissociationconstant lower than 10⁻⁶ M, more preferably lower than 10⁻¹² M and morepreferably lower than 10⁻¹⁵ M, depending on the components structure andthe required needs.

According to an additional aspect of the present invention there isprovided an affinity pair that comprises a cucurbituril, a polyaminestructure being capable of binding thereto and at least one functionalmoiety being attached to the cucurbituril and/or to the polyaminestructure.

The affinity pairs described above can be used, according to the presentinvention, in methods of affinity binding. These methods are effected bycontacting a cucurbituril or a cucurbituril assembly and a polyaminestructure capable of binding thereto.

The contacting is preferably performed under conditions that enableprotonation of the polyamine structure, e.g., acidic conditions.

According to further features in preferred embodiments of the inventiondescribed below, the functional moiety is selected from the groupconsisting of a pharmaceutically active agent, a biomolecule, and alabeling moiety.

A pharmaceutically active agent, according to preferred embodiments ofthe present invention, can be, for example, an anti-proliferative agent,an anti-inflammatory agent, an antibiotic, an anti-hypertensive agent,an antioxidant, a chemotherapeutic agent, an antidepressant, an antihistamine, a vitamin, a hormone, a ligand, an inhibitor, an agonist, anantagonist and a co-factor.

A labeling moiety, according to preferred embodiments of the presentinvention, can be, for example, a probe, a chromophore, a fluorescentcompound, a phosphorescent compound, a heavy metal cluster, and aradioactive labeling compound.

The biomolecule, according to preferred embodiments of the presentinvention can be, for example, an amino acid, a peptide, a protein, anantibody, an antigen, a nucleic acid, a polynucleotide, anoligonucleotide, an antisense, a polysaccharide, a fatty acid, amembrane and a cell.

According to still further features in the described preferredembodiments the at least one functional moiety forms a part of a solidsupport.

According to still further features in the described preferredembodiments the solid support is selected from the group consisting of asurface, a polymer, a resin and a bead.

A representative example of a solid support is a polystyrene resin.

The methods of affinity binding can be applied for a use selected fromthe group consisting of affinity chromatography, affinity labeling,immobilization, bioconjugation, immunohistochemical staining, flowcytometry, in situ hybridization, genetic mapping and immunoassays, asis detailed hereinunder.

Further according to the present invention there is provided a processof isolating at least one cucurbituril from a mixture containing same,which comprises: providing the mixture containing the at least onecucurbituril; providing a column packed with a polyamine structure beingcapable of selectively binding to the at least one cucurbituril; elutingthe mixture through the column, to thereby obtain the column having atleast a first portion of the at least one cucurbituril bound thereto anda first eluent; and releasing the at least first portion of the at leastone cucurbituril from the column having at least the first portion ofthe at least one cucurbituril bound thereto, thereby isolating at leastthe first portion of the at least one cucurbituril from the mixture.

The eluting step above preferably comprises loading the mixture onto thecolumn; and washing the column with a first solvent, the first solventbeing a medium for binding the polyamine structure to the at least onecucurbituril, whereby the releasing step above comprises washing thecolumn with a second solvent, the second solvent being a medium forinterrupting a binding of the polyamine structure to the at least onecucurbituril.

The process may further comprise re-eluting the first eluent through thecolumn, to thereby obtain the column having a second portion of the atleast one cucurbituril bound thereto and a second eluent; releasing thesecond portion of the at least one cucurbituril from the column havingthe second portion of at least one cucurbituril bound thereto, tothereby isolate a second portion of the at least one cucurbituril fromthe mixture; re-eluting the second eluent through the column, to therebyobtain the column having a third portion of the at least onecucurbituril bound thereto and a third eluent; releasing the thirdportion of the at least one cucurbituril from the column having thethird portion of at least one cucurbituril bound thereto, to therebyisolate a third portion of the at least one cucurbituril from themixture; and repeating the re-eluting and the releasing, therebyisolating the at least one cucurbituril from the mixture.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing novel, highly versatile andhighly advantageous synthetic binding pairs of cucurbiturils orcucurbituril assemblies and polyamine structures, which can be used in avariety of applications without being limited by factors such aschemical versatility, stability, irreversibility and the like.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a schematic illustration depicting the main strategies in theapplication of Avidin-Biotin (Av-B) technology;

FIG. 2 is a scheme illustrating a general pathway of synthesizing CB[6];

FIG. 3 is a schematic illustration demonstrating the generation andregeneration of an affinity binding pair used for purification of CB[n]sand rare CB[n]s according to the present invention (Key: a.H₂N(CH₂)₅NHBOC, DMF, Py; b. TFA, CH₂Cl₂ c. Crude product mixtureobtained in the synthesis of dimethylcyclopentano-CB; d. Et₃N, DMF);

FIG. 4 is a scheme illustrating a general pathway for synthesizingdimethylcyclopentano-CB (Compound 4), an exemplary derivatizedcucurbituril according to the present invention;

FIG. 5 is a scheme illustrating a general synthetic pathway forpreparing pentacyclohexano-CB[5] (Compound 10) and an exemplary rarecucurbituril, hexacyclohexano-CB[6] (Compound 11), by reactingcyclohexanoglycoluril (Compound 9) and formaldehyde, and for preparingdecamethylcucurbit[5]uril (Compound 13), and another exemplary rarecucurbituril, dodecamethycucurbit[6]uril (Compound 14), by reactingdimethylglycoluril (Compound 12) and formaldehyde;

FIG. 6 is a scheme illustrating exemplary synthetic pathways forpreparing CB[6] trimers according to the present invention;

FIG. 7 is a scheme illustrating a method of labeling a protein with aprobe, using a double affinity PA-CB[n] pair according to the presentinvention;

FIG. 8 presents the chemical structures and the molecular weights of aPA-CB affinity pair, according to the present invention, compared withan Av-B pair;

FIG. 9 is a scheme illustrating a bifunctional CB[6] binder according tothe present invention;

FIG. 10 presents the FTIR spectrum of chloromethylated polystyrene beads(Compound 5) according to the present invention;

FIG. 11 presents the FTIR spectrum of Boc-protected aminated polystyrenebeads (Compound 6) according to the present invention;

FIG. 12 presents the FTIR spectrum of the fully protonated aminatedpolystyrene beads (Compound 7) according to the present invention;

FIG. 13 presents the FTIR spectrum of the fully protonated aminatedpolystyrene beads (Compound 7) bound to a CB[6] (Compound 1) accordingto the present invention;

FIG. 14 presents the ¹H NMR spectrum of a mixture of Compound 4 andCompound 1, obtained and isolated by an affinity chromatographyaccording to the present invention;

FIG. 15 presents the ESI-MS spectrum of a mixture of Compound 4 andCompound 1 obtained and isolated by an affinity chromatography accordingto the present invention;

FIG. 16 presents the ¹H NMR spectrum of hexacyclohexano-CB[6] (Compound11), obtained and isolated by an affinity chromatography according tothe present invention;

FIG. 17 presents the ¹³C NMR spectrum of hexacyclohexano-CB[6] (Compound11), obtained and isolated by an affinity chromatography according tothe present invention;

FIG. 18 presents the ESI-MS spectrum of hexacyclohexano-CB[6] (Compound11) obtained and isolated by an affinity chromatography according to thepresent invention;

FIG. 19 presents the ¹H-NMR spectrum of dodecamethycucurbit[6]uril(Compound 14) obtained and isolated by an affinity chromatographyaccording to the present invention;

FIG. 20 presents the ¹³C-NMR spectrum of dodecamethycucurbit[6]uril(Compound 14) obtained and isolated by an affinity chromatographyaccording to the present invention;

FIG. 21 presents the ESI-MS spectrum of dodecamethycucurbit[6]uril(Compound 14) obtained and isolated by an affinity chromatographyaccording to the present invention;

FIG. 22 is a scheme illustrating a general synthetic pathway forpreparing derivatized glycolurils according to the present invention;

FIG. 23 is a scheme illustrating another general pathway forsynthesizing derivatized cucurbiturils according to a preferredembodiment of the present invention, an a synthetic pathway forpreparing a CB trimer therefrom and such a CB having a protein attachedthereto;

FIG. 24 is a scheme illustrating an exemplary synthetic pathway forpreparing a linear cucurbituril trimer according to a preferredembodiment of the present invention;

FIG. 25 is a scheme illustrating a synthetic pathway for preparing acucurbituril trimer having a rigid assembling unit according to apreferred embodiment of the present invention;

FIG. 26 is a scheme illustrating a synthetic pathway for preparing anexemplary polyamine structure according to the present invention, havinga rigid threading moiety and an affinity pair thereof with a CB[6],according to a preferred embodiment of the present invention;

FIG. 27 is a scheme illustrating a synthetic pathway for preparinganother exemplary polyamine structure according to the presentinvention, having a rigid threading moiety;

FIG. 28 is a scheme illustrating a synthetic pathway for preparingadditional exemplary polyamine structures according to the presentinvention, having a rigid threading moiety (Compound 53 and Compound55);

FIG. 29 is a scheme illustrating a synthetic pathway for preparinganother exemplary polyamine structure according to the presentinvention, having a rigid threading moiety (Compound 59);

FIG. 30 is a scheme illustrating a synthetic pathway for preparing anexemplary polyamine structure according to the present invention, havinga rigid threading moiety (Compound 62);

FIG. 31 is a scheme illustrating a synthetic pathway for preparinganother exemplary polyamine structure according to the presentinvention, having a rigid threading moiety (Compound 68); and

FIGS. 32 a-b present the top view (FIG. 32 a) and the side view (FIG. 32b) of the solid-state structure of an affinity pair Compound 46 withCB[6], as obtained from X-ray crystallography measurements (all hydrogenatoms are omitted and dashed lines indicate hydrogen bond).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of novel synthetic affinity binding pairs ofcucurbiturils and polyamines, which can be used in various purification,detection and therapeutic applications. Specifically, the presentinvention is of (i) novel cucurbituril assemblies; (ii) polyaminestructures that are designed capable of binding to the cucurbiturilassemblies; (iii) affinity binding pairs which comprise the polyaminestructures and the cucurbituril assemblies of the present invention; and(iv) use of these affinity pairs in various applications such as, butnot limited to, isolation and purification of biological molecules viaaffinity chromatography, immunohistochemical staining, introducingmultiple labels into tissues, localizing hormone binding sites, flowcytometry, in situ localization and hybridization, radio-, enzyme-, andfluorescent immunoassays, neuronal tracing, genetic mapping, hybridomascreening, purification of cell surface antigens, coupling of antibodiesand antigens to solid supports, examination of membrane vesicleorientation, and drug delivery.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Affinity pairs serve as a basis for the development of many fundamentalresearch, industrial tools and techniques in fields such as chemistry,biology and medicine. One of the presently most utilized affinity pairis the Avidin-Biotin affinity pair (Av-B). As is discussed in detailhereinabove, avidin is an egg-white derived protein, which can bind atleast one, and up to four, biotin molecules. However, although the Av-Baffinity pair is characterized by a high affinity binding constant,K_(D)=10⁻¹⁵ M [Wilchek M, Methods Enzymol., entire Volume 184, 1990],this system suffers several disadvantages, which severely limit its use,as is discussed in detail hereinabove.

Thus, for example, an Av-B system is limited by the non-specific andnon-controllable number of binding sites on the avidin part (four), thesensitive protein nature of the avidin part, a limited chemistry thatcan be applied in these systems, an inflexible binding constant and anirreversible binding, as well as other physical, chemical and biologicalconstraints, as is detailed hereinabove and is further discussedhereinbelow.

Due to these limitations, the Av-B system cannot be efficientlyutilized, for example, in applications that require organic media (e.g.,purification of organic mixtures, removal of contaminants), versatilechemical interactions (e.g., binding to therapeutic moieties that haveversatile functional groups), multiple labeling (e.g., when substantialsignal amplification should be applied), light measurements (e.g.,detection of peptides signals that are obscured by avidin), andcontrollable and/or reversible binding (e.g., affinity chromatography).

In a search for novel affinity pairs that would be devoid of suchlimitations and could therefore be efficiently utilized in a wider rangeof applications, the present inventor have envisioned that by utilizingthe unique host-guest properties of cucurbiturils and polyamines, highlyefficient affinity binding systems, characterized by e.g., higherstability, chemical versatility and controllable binding, could beobtained.

As is described hereinabove, cucurbiturils (CBs) are macrocycliccavitand compounds comprised of several glycoluril units andcharacterized by a hydrophobic cavity that is accessible through twoidentical, polar, carbonyl-fringed portals.

Herein, substituted and unsubstituted CBs, including derivatives andhomologues thereof, are collectively referred to as CB[n], whereby nrepresents the number of glycoluril units in the CB.

The rigid structure and the combination of a hydrophobic cavity withpolar portals allow the CB[n]s to act as synthetic selective receptorsof various cations. The selectivity of a CB[n] toward specific cationsstems primarily from its size, i.e., the number of glycoluril units (n)composing the CB[n].

However, the number of glycoluril units in a formed CB has been shown tofurther depend on the nature of the glycoluril building unit, whichaffects the thermodynamic stability of the formed CB. Hence,cucurbiturils formed from unsubstituted glycolurils typically includeCB[6] as the major product, whereby cucurbiturils formed fromsubstituted glycolurils typically include CB[5] as the major product(Day et al., Molecules, 2003, 8, 74-84).

The most strong and efficient affinity binding to CB[n]s has beenobserved with polyammonium ions, henceforth referred to as polyamines(PAs). The PA threads the CB[n] through the cavity with what is referredto in the art and is further defined hereinbelow as a threading moiety.

It has been shown that the recognition interactions between apolyammonium ion and a CB depend on the structure and chemical nature ofthe PA, and its suitability to the size of the CB[n], as is detailedhereinbelow. The strongest affinity of PA to CB[6] has been observedwith n-alkyldiammonium ions, whereby the optimal chain length was foundto be 5-6. The resulting dissociation constants for such PA-CB pairswere found to be in the micro-molar range (Mock, W. L. in ComprehensiveSupramolecular Chemistry; Vögtle, F., Ed.; Elsevier Press: New York,1996; Vol. 2, pp 477493).

To date, studies of the interactions between CB[n] and PA have beenperformed only with a single pair of one cucurbituril and one polyaminein solution, or several unconnected cucurbiturils with a polyamine,teaching the utilization of these interactions in applications such ascatalysis of dipolar cycloadditions (Mock et al., J. Org. Chem. 1983,48, 3920-3619, and 1989, 54, 5308-5302) as a molecular bead in molecularnecklaces and polyrotaxanes (Whang et al. J. Am. Chem. Soc. 1998, 120,4899-4900 and Kim, K. Chem. Soc. Rev. 2002, 31, 96-107) molecular bowls(Jeon et al. J. Am. Chem. Soc. 1996, 118, 9790-9791) and for the removalof contaminants from aqueous waste streams (Kornmuller et al. Water Res.2001, 35, 3317-3324). Yet no reports have been made where the PA-CBinteraction is used as an affinity pair for purification, detection andtherapeutic applications.

While conceiving the present invention, it was envisioned that thehighly specific recognition of a PA-CB pair, the diverse chemistry thecan be applied while preparing and utilizing these compounds, andparticularly, the cumulative nature of the binding strength when morethan one pair of PA-CB are acting synchronously, could be utilized forpreparing and practicing a novel affinity pair system that would besuperior to the presently used Av-B system.

Thus, it was envisioned that unlike the Av-B affinity pair, a PA-CBaffinity pair system would allow multiplying the binding strength of asingle PA-CB pair when two, three or more affinity pairs are actingsimultaneously, and would further allow fashioning a PA-CB affinity pairwith almost any desired binding constant.

More specifically, the present inventor have envisioned that bydesigning suitable cucurbituril derivatives, synthetic cucurbiturilassemblies, characterized by unique, predetermined, spatial spread andcontents of CB[n]s in a predefined relative positioning in space, couldbe obtained. It was further envisioned that suitable polyaminestructures counterparts could be designed correspondingly, exhibiting aunique number and type of threading moieties at matching spatial spreadas to interact with a specific cucurbituril assembly. It was thusfurther envisioned that such cucurbituril assemblies and theircounterpart polyamine structures would allow the fabrication of highlyefficient affinity pairs, characterized by highly beneficial andadvantageous chemical and physical properties.

Thus, while designing a CB-PA affinity pair according to the presentinvention, cucurbiturils having one or more functional groups, which arealso referred to herein interchangeably as derivatized cucurbiturils,and which are aimed at forming cucurbituril assemblies, have beendesigned.

Each of these cucurbiturils, according to the present invention,therefore has a functional group, which, upon reaction, either directlyor indirectly, with one or more derivatized cucurbituril, forms acucurbituril assembly, as is defined hereinbelow.

As is discussed above, CBs are composed of a number of glycoluril units,represented by n in CB[n], which upon reaction with formaldehydes, formthe cucurbituril.

The CBs according to the present invention preferably have between 5 and20 glycoluril units, such that n ranges from 5 to 20. More preferably,the CBs according to the present invention have between 5 and 10glycoluril units, such that n ranges from 5 to 10. More preferably, theCBs according to the present invention have between 5 and 8 glycolurilunits, and therefore include CB[5], CB[6], CB[7] and CB[8]. Morepreferably, the CBs according to the present invention include CB[5]and/or CB[6], with CB[6]s being the most preferred cucurbiturilsexhibiting the highest binding affinity to polyamines (Mock, W. L. inComprehensive Supramolecular Chemistry; Vögtle, F., Ed.; Elsevier Press:New York, 1996; Vol. 2, pp 477-493, which is incorporated by referenceas if fully set forth herein).

As is further discussed hereinabove, the size of the formed cucurbiturilis determined by the nature of the glycoluril units, which affects thethermodynamic stability of the reaction intermediates and hence theirproducts distribution. Thus, typically, CBs are formed as a mixture ofthermodynamically favorable and unfavorable products as the major andminor products, respectively. Particularly, cucurbiturils formed fromunsubstituted glycolurils typically include CB[6] as the major product,whereby cucurbiturils formed from substituted glycolurils typicallyinclude CB[5] as the major product (Day et al., Molecules, 2003, 8,74-84).

As used herein, the phrase “functional group” describes a chemicalmoiety that is capable of undergoing a chemical reaction that typicallyleads to a bond formation. The bond, in the case of the presentinvention, can be a covalent bond, a ionic bond, a hydrogen bond and thelike and is preferably a covalent bond. Chemical reactions that lead toa bond formation include, for example, nucleophilic and electrophilicsubstitutions, nucleophilic and electrophilic addition reactions,elimination reactions, cycloaddition reactions, rearrangement reactionsand any other known organic reactions that involve a functional group.

Representative examples of suitable functional groups according to thepresent invention include, without limitation, amine, halide, sulfonate,sulfinyl, phosphonate, phosphate, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, carboxylate,thiocarbamate, urea, thiourea, carbamate, C-amide, N-amide, guanyl,guanidine and hydrazine, as these terms are defined hereinafter.

As used herein, the term “amine” refers to a —NR′R″ group, wherein R′and R″ are each independently hydrogen, alkyl, cycloalkyl, aryl, asthese terms are defined hereinbelow. The amine group can therefore be aprimary amine, where both R′ and R″ are hydrogen, a secondary amine,where R′ is hydrogen and R″ is alkyl, cycloalkyl or aryl, or a tertiaryamine, where each of R′ and R″ is independently alkyl, cycloalkyl oraryl.

The term “alkyl” refers to a saturated aliphatic hydrocarbon includingstraight chain and branched chain groups. Preferably, the alkyl grouphas 1 to 20 carbon atoms. Whenever a numerical range; e.g., “1-20”, isstated herein, it implies that the group, in this case the alkyl group,may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up toand including 20 carbon atoms. More preferably, the alkyl is a mediumsize alkyl having 1 to 10 carbon atoms. Most preferably, unlessotherwise indicated, the alkyl is a lower alkyl having 1 to 4 carbonatoms. The alkyl group may be substituted or unsubstituted. Whensubstituted, the substituent group can be, for example, hydroxyalkyl,trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,heteroalicyclic, amine, halide, sulfonate, sulfinyl, phosphonate,phosphate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,thioaryloxy, cyano, nitro, azo, sulfonamide, carboxylate, thiocarbamate,urea, thiourea, carbamate, C-amide, N-amide, guanyl, guanidine andhydrazine.

The term “cycloalkyl” refers to an all-carbon monocyclic or fused ring(i.e., rings which share an adjacent pair of carbon atoms) group whereone or more of the rings does not have a completely conjugatedpi-electron system. The cycloalkyl group may be substituted orunsubstituted. When substituted, the substituent group can be, forexample, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfinyl,phosphonate, phosphate, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, carboxylate,thiocarbamate, urea, thiourea, carbamate, C-amide, N-amide, guanyl,guanidine and hydrazine.

The term “aryl” refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. The aryl groupmay be substituted or unsubstituted. When substituted, the substituentgroup can be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide,sulfonate, sulfinyl, phosphonate, phosphate, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide,carboxylate, thiocarbamate, urea, thiourea, carbamate, C-amide, N-amide,guanyl, guanidine and hydrazine.

The term “halide” group refers to fluorine, chlorine, bromine or iodine.

The term “sulfonate” refers to an —S(═O)₂—R′ group, where R′ is asdefined herein.

The term “sulfinyl” refers to a —S(═O)—R′ group, where R′ is as definedherein.

The term “phosphonate” describes a —P(═O)(OR′)(OR″) group, with R′ andR″ as defined herein.

The term “phosphate” describes a —O—P(═O)(OR′)(OR″) group, with R′ andR″ as defined herein.

The term “hydroxyl” refers to a —OH group. The term “alkoxy” refers toboth an —O-alkyl and an —O-cycloalkyl group, as defined herein.

The term “aryloxy” refers to both an —O-aryl and an —O-heteroaryl group,as defined herein.

The term “thiohydroxy” refers to an —SH group

The term “thioalkoxy” refers to both an —S-alkyl group, and an—S-cycloalkyl group, as defined herein.

The term “thioaryloxy” refers to both an —S-aryl and an —S-heteroarylgroup, as defined herein.

The term “cyano” refers to a —C≡N group.

The term “nitro” refers to an —NO₂ group.

The term “azo” refers to a —N═NR′ group, with R′ as defined hereinabove.

The term “sulfonamide” refers to an —S(═O)₂—NR′R″ for S-sulfonamidegroup, and to an —NR′S(═O)₂—R″ for N-sulfonamide group, with R′ and R″as defined herein.

The term “carboxylate” refers to a —C(═O)—OR′ group, where R′ is asdefined herein.

The term “carbamate” refers to a —OC(═O)—NR′R″ for O-carbamate group,and to a to R″OC(═O)—NR′— for N-carbamate group, with R′ and R″ asdefined herein.

The term “thiocarbamate” refers to an —SC(═O)—NR′R″ for O-thiocarbamategroup, and to an R″SC(═O)NR′— for N-thiocarbamate group, with R′ and R″as defined herein.

The term “urea” and/or “ureido” refers to a —NR′C(═O)—NR″R′″ group,where R′ and R″ are as defined herein and R′″ is defined as either R′ orR″.

The term “thiourea” and/or “thioureido” refers to a —NR′—C(═S)—NR′R′″group, with R′, R″ and R′″ as defined herein.

The term “C-amide” refers to a —C(═O)—NR′R″ group, where R′ and R″ areas defined herein.

The term “N-amide” refers to a R′C(═O)—NR″— group, where R′ and R″ areas defined herein.

The term “guanyl” refers to a R′R″NC(═N)— group, where R′ and R″ are asdefined herein.

The term “guanidine” refers to a —R′NC(═N)—NR″R′″ group, where R′ and R″are as defined herein and R′″ is defined as either R′ or R″.

The term “hydrazine” refers to a —NR′—NR″R′″ group, with R′, R″ and R′″as defined herein.

The functional group can be attached to the cucurbituril either directlyor indirectly via a spacer.

The spacer can be, for example, an alkyl having 1 to 20 carbon atoms, analkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to20 carbon atoms, an alkoxy having 1 to 20 carbon atoms, an aminoalkylhaving 1 to 20 carbon atoms, a cycloalkyl having 5 to 20 atoms, aheteroalicyclic having 4 to 20 carbon atoms, an aryl having 6 to 20carbon atoms and a heteroaryl having 6 to 20 carbon atoms, as theseterms are defined hereinabove.

The term “heteroaryl” refers to a monocyclic or fused ring (i.e., ringswhich share an adjacent pair of atoms) group having in the ring(s) oneor more atoms, such as, for example, nitrogen, oxygen and sulfur and, inaddition, having a completely conjugated pi-electron system. Examples,without limitation, of heteroaryl groups include pyrrole, furane,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine,quinoline, isoquinoline and purine. The heteroaryl group may besubstituted or unsubstituted. When substituted, the substituent groupcan be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl,alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate,sulfinyl, phosphonate, phosphate, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, carboxylate,thiocarbamate, urea, thiourea, carbamate, C-amide, N-amide, guanyl,guanidine and hydrazine. Representative examples are pyridine, pyrrole,oxazole, indole, purine and the like.

The term “heteroalicyclic” refers to a monocyclic or fused ring grouphaving in the ring(s) one or more atoms such as nitrogen, oxygen andsulfur. The rings may also have one or more double bonds. However, therings do not have a completely conjugated pi-electron system. Theheteroalicyclic may be substituted or unsubstituted. When substituted,the substituent group can be, for example, hydroxyalkyl, trihaloalkyl,cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine,halide, sulfonate, sulfinyl, phosphonate, phosphate, hydroxy, alkoxy,aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo,sulfonamide, carboxylate, thiocarbamate, urea, thiourea, carbamate,C-amide, N-amide, guanyl, guanidine and hydrazine. Representativeexamples are piperidine, piperazine, tetrahydrofurane, tetrahydropyrane,morpholino and the like.

Preferably the spacer is a medium size spacer such as an alkyl having 1to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, analkynyl group having 2 to 10 carbon atoms, an alkoxy having 1 to 10carbon atoms, an aminoalkyl having 1 to 10 carbon atoms, a cycloalkylhaving 5 to 10 atoms, a heteroalicyclic having 4 to 10 carbon atoms, anaryl having 6 to 12 carbon atoms and a heteroaryl having 6 to 12 carbonatoms.

More preferably, the spacer is a lower size spacer such as an alkylhaving 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms,an alkynyl group having 2 to 6 carbon atoms, an alkoxy having 1 to 6carbon atoms, an aminoalkyl having 1 to 6 carbon atoms, a cycloalkylhaving 5 to 8 atoms, a heteroalicyclic having 4 to 8 carbon atoms, anaryl having 6 to 12 carbon atoms and a heteroaryl having 6 to 12 carbonatoms.

Each of the spacers described above can be substituted or unsubstituted.When substituted, the substituent can be, for example, hydroxyalkyl,trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,heteroalicyclic, amine, halide, sulfonate, sulfinyl, phosphonate,phosphate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,thioaryloxy, cyano, nitro, azo, sulfonamide, carboxylate, thiocarbamate,urea, thiourea, carbamate, C-amide, N-amide, guanyl, guanidine andhydrazine, as these terms are defined hereinabove.

Each of the spacers described above can optionally be interrupted by oneor more heteroatoms such as, for example, oxygen, sulfur, nitrogen,phosphor and the like.

Thus, the spacer can be a saturated or unsaturated hydrocarbon chain,optionally interrupted by e.g., oxygen, sulfur or nitrogen heteroatoms.The spacer can further be such a hydrocarbon chain, which is linkedeither to the cucurbituril or to the functional group via an aminogroup, as, for example, in the case of an aminoalkyl spacer and analkoxy spacer.

Whenever the spacer is a cyclic spacer, e.g., cycloalkyl or aryl, it canbe either linked to the cucurbituril via a single ring atom or fused tothe cucurbituril via two or more ring atoms.

Whenever the spacer is a heterocyclic spacer, e.g., a heteroalicyclic ora heteroaryl, the functional group can be incorporated within thespacer, namely, as a substituted or unsubstituted heteroatom.

In a representative example, the functional group is a secondary aminethat forms a part of a nitrogen-containing heteroalicyclic spacer, e.g.,pyrrolidine, which is fused to the cucurbituril via two ring atoms, asis shown, for example, in FIG. 6, Compound 20.

The derivatized cucurbituril described above can be represented by thegeneral formula that follows:

As is described hereinabove, n preferably ranges from 5 to 20, morepreferably from 5 to 10, more preferably from 5 to 8 and most preferablyfrom 5 to 6.

The atoms represented by X₁, X₂ . . . X_(n) and E₁, E₂ . . . E_(n) arederived from the glycoluril units that form the derivatized cucurbituriland thus can be for example O, S and NR′. Preferably, each of X₁, X₂ . .. X_(n) and E₁, E₂ . . . E_(n) is oxygen (O).

The substituents at positions R′, A₁, A₂ . . . A_(n), D₁, D₂ . . .D_(n), Y₁, Y₂ . . . Y_(n) and Z₁, Z₂ . . . Z_(n) can be, for example,hydrogen, an alkyl having 1 to 20 carbon atoms, an alkenyl group having2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, analkoxy having 1 to 20 carbon atoms, an aminoalkyl having 1 to 20 carbonatoms, a cycloalkyl having 5 to 20 atoms, a heteroalicyclic having 4 to20 carbon atoms, an aryl having 6 to 20 carbon atoms and a heteroarylhaving 6 to 20 carbon atoms.

Y₁, Y₂ . . . Y_(n) and Z₁, Z₂ . . . Z_(n) are typically derived from thealdehyde units that form the cucurbituril and therefore preferablyeither Y₁, Y₂ . . . Y_(n) or Z₁, Z₂ . . . Z_(n) are hydrogen atoms andmore preferably all of Y₁, Y₂ . . . Y_(n) and Z₁, Z₂ . . . Z_(n) arehydrogen atoms.

Since the derivatized cucurbituril according to the present invention isdesigned capable of forming a cucurbituril assembly, it includes atleast one functional group, as is described is detail hereinabove, suchthat in the formula above, one or more of A₁, A₂ . . . A_(n) and D₁, D₂,. . . D_(n) comprises the functional group. The functional group can beattached to the cucurbituril backbone either directly or indirectly viaa spacer, such that one or more of A₁, A₂ . . . A_(n) and D₁, D₂, . . .D_(n) is a functional group per se or a spacer, as is detailedhereinabove, terminated by the functional group.

In a representative example, a derivatized cucurbituril according to thepresent invention has the general formula above, wherein n is 6, each ofX₁, X₂ . . . X₆ and E₁, E₂ . . . E₆ is oxygen, each of Y₁, Y₂ . . . Y₆and Z₁, Z₂ . . . Z₆ is hydrogen and one of A₁, A₂ . . . A₆ and D₁, D₂, .. . D₆ is a secondary amine that forms a part of a nitrogen-containingheteroalicyclic spacer, e.g., pyrrolidine, which is fused to thecucurbituril at the A_(n) and D_(n) positions, as is shown in FIG. 6(Compound 20).

As stated hereinabove, the functional group in the derivatizedcucurbiturils of the present invention is aimed at forming acucurbituril assembly.

As used herein, the phrase “cucurbituril assembly” describes a molecularstructure that includes at least two cucurbituril units covalentlyattached one to the other and forming, depending on the number ofcucurbituril units and the assembly's structure, either a dimer, atrimer, a polymer, an oligomer, a dendrimer or a cluster of thecucurbiturils.

As used herein the term “dimer”, which is also referred to hereininterchangeably as “bis-cucurbituril structure”, describes an assemblyof two cucurbiturils being covalently linked one to the other.

As used herein the term “trimer”, which is also referred to hereininterchangeably as “bis-cucurbituril structure”, describes an assemblyin the form of a branched or linear chain of three cucurbiturils.

As used herein the terms “polymer” and “oligomer” describe an assemblyin the form of a long (more than 10) or short (of between 4 and 10),linear or branched chain of cucurbiturils, respectively.

As used herein the term “dendrimer” describes a perfectlycascade-branched, highly defined structure having a core and an interiorarea containing branch upon branch of repeat units or generations withradial connectivity to the core, and an exterior or surface region ofterminal moieties attached to the outermost generation and to aplurality of cucurbiturils.

The term “cluster” describes an undefined structure of a plurality ofcucurbiturils.

The functional group in each of the derivatized cucurbiturils that formsa part of the cucurbituril assembly serves for covalently binding thecucurbiturils one to another, so as to form the assembly.

In a preferred embodiment of the present invention, the cucurbiturilassembly comprises one or more assembling unit(s), each being covalentlyattached to each cucurbituril in the assembly, directly or indirectly,via the functional group.

As used herein, the phrase “assembling unit” describes a chemical moietythat links two or more cucurbiturils via one or more covalent bonds. Ingeneral, the assembling unit can be formed during the assemblyformation, such that by reacting the functional groups of the two ormore cucurbiturils, the assembling unit is formed as a new chemicalentity, or can be an independent chemical moiety that have two or morereactive sites or groups to which the functional groups of thecucurbituril can be attached, either directly or indirectly, as isdetailed hereinunder.

Thus, for example, in cases where the cucurbituril assembly is a dimerof two derivatized cucurbiturils according to the present invention, theassembling unit can be a chemical moiety to which each of thederivatized cucurbiturils is attached via the functional group.

In cases where the cucurbituril assembly is a branched trimer, theassembling unit can be either a chemical moiety to which each of thethree derivatized cucurbiturils is attached via their functional groups.In cases where the cucurbituril assembly is a linear trimer, theassembly comprises two assembling units, each being a chemical moiety towhich two derivatized cucurbiturils is attached via their functionalgroups.

In cases where the cucurbituril assembly is an assembly of fourderivatized cucurbiturils, there may be, for example, two types ofassembling units in one assembly, such that one assembling unit is achemical moiety to which each of two derivatized cucurbiturils isattached via the functional group, and of the other assembling unit is achemical moiety to which each of the two assembling units of the firstclass is attached, so as to form “a dimer of dimers”. A representativeexample of such an assembly is illustrated in FIG. 9.

In cases where the cucurbituril assembly is a dendrimer, the assemblingunit can be the core, the interior area and the exterior layer ofterminal moieties to which a plurality of derivatized cucurbiturils isattached via their functional group.

By designing versatile derivatized cucurbiturils having versatilefunctional groups and versatile yet suitable assembling units, versatilecucurbituril assemblies can be formed, in a predetermined fashion, usingsimple and convenient chemical reactions.

A cucurbituril assembly according to the present invention, can thus beformed via one or more of the following exemplary, non-limitingpathways:

(i) Two derivatized CB[n]s according to the present invention, eachhaving a different functional group, are reacted so as to form acovalent bond between the two functional groups directly. In this case,the formed bond represents the assembling unit.

In a representative example, a derivatized cucurbituril having an aminegroup as a functional group (the amine group is either directly orindirectly attached to the CB[n]), is reacted with a derivatizedcucurbituril having a carboxylic acid as a functional group (thecarboxylic acid group is either directly or indirectly attached to theCB[n]), to thereby form an amide bond as the assembling unit of acucurbituril dimer.

(ii) Three or more derivatized cucurbiturils according to the presentinvention, are reacted as above, so as to form a linear or branchedtrimer, oligomer or polymer of cucurbiturils. In this case, each of thederivatized cucurbiturils has at least two functional groups, and thefunctional groups are designed so as to react one with the other, tothereby form the assembling unit(s). In a representative example,derivatized cucurbiturils according to the present invention that havetwo methyl ester groups as functional groups are reacted with ethyleneglycol, so as to produce a variety of cucurbituril polymers having aplurality of ethyl esters or diethyl esters as the assembling units. Inanother example, derivatized cucurbiturils having as functional groupscarboxylic acid and/or amine are reacted so as to produce a linear orbranched trimer, oligomer or polymer of cucurbiturils having a pluralityof amide bonds as the assembling units is formed.

(iii) The functional groups of two or more a derivatized cucurbiturilscan be converted to an assembling unit, or, alternatively, attached toan assembling unit being an independent chemical entity, andsuccessively one or more additional derivatized cucurbituril areattached to the assembling unit via their functional groups thus forminga cucurbituril assembly.

(iv) The functional groups of two or more a derivatized cucurbiturilsare converted simultaneously to functional groups which, when reactedtogether, form an assembling unit and a cucurbituril assembly. In arepresentative example, which is further illustrated in FIG. 6 and isdescribed in detail in Example 6 in the Examples section that follows,the thus formed assembling unit is a triazine ring being formed by firstconverting an amine functional group of derivatized cucurbiturils tocyanoamine, and then allowing three cyanoamine-derivatized cucurbiturilunits to form the triazine assembling unit and thus to form acucurbituril trimer.

(v) The assembling unit is provided, or is independently formed first,and successively two or more derivatized cucurbiturils are attachedthereto either by reacting the functional groups of the derivatizedcucurbiturils with the assembling unit or by providing an assemblingunit that is substituted by two or more glycoluril units and reactingthe latter with aldehydes. A general representative procedure of thelatter in described in detail in the Examples section that follows.

(vi) A cucurbituril assembly of derivatized cucurbiturils according tothe present invention is formed first in one or more of the paths i-vhereinabove, and a plurality of these assemblies are attached to form anassembly of cucurbituril assemblies via one or more assembling unit thusforming a dendrimer or cluster of CB[n] in one or more of the paths i-vhereinabove. A representative example of a dimer of CB[n] dimers isillustrated in FIG. 9, wherein the assembling unit in each CB[n] dimeris a triazine ring, and the assembling unit of the dimer of dimmers is aderivative of trimesic acid (benzene-1,3,5-tricarboxylic acid).

The assembling unit according to the present invention can therefore becomprised of one or more subunits that are covalently attached one toanother. Representative examples of such subunits include, withoutlimitation, cycloalkyl, heteroalicyclic, aryl, heteroaryl, polyaryl,polyheteroaryl, amide, sulfonamide, phosphonate, phosphate, carboxyl,thiocarboxyl, carbamyl, thiocarbamyl, ureido, thioureido, and hydrazine.

Cyclic subunits such as, for example, cycloalkyl, heteroalicyclic, aryl,heteroaryl, polyaryl, and polyheteroaryl, typically serve, according tothe present invention, as chemical moieties to which two or morederivatized cucurbiturils are attached via the functional groups. Thesecyclic subunits, when being fused one to another while forming anassembling unit according to the present invention, provide anassembling unit that is characterized by a rigid structure, as isillustrated, for example, in FIGS. 24 and 25. Such a rigid assemblingunit is highly advantageous, as is discussed hereinbelow.

Thus, as used in this context of the present invention, the termcycloalkyl, describes a cycloalkyl, as is defined hereinabove, to whichtwo or more derivatized cucurbiturils can be attached via theirfunctional groups. Since unsubstituted cycloalkyls are typically notchemically reactive, preferred cycloalkyls that are usable in thiscontext of the present invention include cycloalkyls that aresubstituted by at least one substituent as is described hereinabove.

Similarly, the term “heteroalicyclic” as used in this context of thepresent invention, describes a heteroalicyclic, as is definedhereinabove, to which two or more derivatized cucurbiturils can beattached via their functional groups. The functional groups can beattached to the one or more heteroatoms of an unsubstitutedheteroalicyclic and/or to one or more substituents of a substitutedheteroalicyclic as described hereinabove.

The term “aryl” as used in this context of the present invention,describes an aryl, as is defined hereinabove, to which two or morederivatized cucurbiturils can be attached via their functional groups.The functional groups can be attached to one or more positions of anunsubstituted aryl and/or to one or more positions of a substitutedheteroalicyclic as described hereinabove. Typically, substituted arylsare more susceptible to chemical reactions than unsubstituted aryls andare therefore preferably used in this context of the present invention.

The term “heteroaryl” as used in this context of the present invention,describes a heteroaryl, as is defined hereinabove, to which two or morederivatized cucurbiturils can be attached via their functional groups. Arepresentative example of a heteroaryl that is highly usable in thiscontext of the present invention is triazine (as is described, forexample, in the Examples section that follows and is further illustratedin FIGS. 5-9).

The term “polyaryl” as used in this context of the present invention,describes a structure of two or more fused aryl rings, as is definedhereinabove, to which two or more derivatized cucurbiturils can beattached via their functional groups. The functional groups can beattached to one or more positions of an unsubstituted polyaryl asdescribed hereinabove. Non-limiting examples of polyaryls are pentalene,indene, naphthalene, anthracene, pyrene, triphenylene, phenalene andcoronene. Typically, substituted polyaryls are more susceptible tochemical reactions than unsubstituted polyaryls and are thereforepreferably used in this context of the present invention. Arepresentative example of a polyaryl that is highly usable in thiscontext of the present invention is coronene (as is described, forexample, in the Examples section that follows and is further illustratedin FIG. 24).

The term “polyheteroaryl” as used in this context of the presentinvention, describes a structure of two or more fused heteroaryl, as isdefined hereinabove, to which two or more derivatized cucurbiturils canbe attached via their functional groups. Non-limiting examples ofpolyheteroaryls are quinoline, benzophenanthroline, phenazine andtriazaphenalene.

Other subunits such as, for example, amide, sulfonamide, carboxyl,thiocarboxyl, carbamyl, thiocarbamyl ureido, thioureido, and hydrazine,are typically formed as a product of the reaction of two functionalgroups, as is outlined hereinabove. The assembling unit can include oneof these exemplary subunits, or any combination thereof.

The term “amide” as used in this context of the present invention,refers to a —C(═O)NR*— group where R* can be hydrogen, alkyl,cycloalkyl, aryl, another assembling unit or a cucurbituril.

The term “sulfonamide” as used in this context of the present invention,refers to a —S(═O)₂—NR*— group where R* is as defined hereinabove.

The term “carboxyl” as used in this context of the present invention,refers to a —C(═O)—O— group.

The term “thiocarboxyl” as used in this context of the presentinvention, refers to a —C(═O)—S— group.

The term “carbamyl” as used in this context of the present invention,refers to a —OC(═O)—NR*— group where R* is as defined hereinabove.

The term “thiocarbamyl” as used in this context of the presentinvention, refers to a —SC(═O)—NR*— group where R* is as definedhereinabove.

The term “ureido” as used in this context of the present invention,refers to a —NR*C(═O)—NR**— group where R* is as defined hereinabove andR** is as defined for R*.

The term “thioureido” as used in this context of the present invention,refers to a —NR*C(═S)—NR**— group where R* and R** are as definedhereinabove.

The term “hydrazine” as used in this context of the present invention,refers to a —NR*—NR**— group where R* and R** are as definedhereinabove.

Hence, according to the present invention, by selecting the derivatizedcucurbiturils, the assembling units and/or the assembly formationpathway, versatile cucurbituril assemblies having predefined spatialspread and contents, namely, predefined CB[n]s units positioned in apredefined relative positioning in space can be produced.

As is outlined hereinabove, the present inventor have envisioned that bydesigning polyamine structures that are structurally and chemicallysuitable to affinity bind to these assemblies, the strong and versatileinteractions between polyamine and cucurbiturils could be beneficiallyutilized so as to achieve cumulative and cooperative binding when two ormore PA-CB affinity pairs are acting synchronously.

Hence, in order to provide such PA-CB affinity pairs, polyaminestructures capable of binding to one or more cucurbiturils and,preferably, to a cucurbituril assembly according to the presentinvention, have been designed.

As is discussed in detail hereinabove, it has been shown that theinteractions between a PA and a CB involve threading of the PA into theCB[n] through the cavity. It has been further shown that the recognitioninteractions between a polyammonium ion and a CB depend on the structureand chemical nature of the PA, and its suitability to the size of theCB[n]. Thus, for example, the strongest affinity of PA to CB[6] has beenobserved with n-alkyldiammonium ions, whereby the optimal chain lengthwas found to be 5-6. The resulting dissociation constants for such PA-CBpairs were found to be in the micro-molar range the strength of theseinteractions depend on both the length and chemical nature of thepolyamine (Mock, W. L. in Comprehensive Supramolecular Chemistry;Vögtle, F., Ed.; Elsevier Press: New York, 1996; Vol. 2, pp 477-493).

Hence, each of these polyamine structures, according to the presentinvention, comprises two or more amine groups and one or more threadingmoiety which is terminated or interrupted by the amine groups, and issuitably sized to the cucurbituril assembly.

As used herein, the phrase “polyamine structure” describes a compoundthat comprises two or more amine groups. The compound can be comprisedof a hydrocarbon skeleton, e.g., substituted and/or unsubstitutedalkyls, cycloalkyls, aryls and combinations thereof, and can optionallybe interrupted by one or more heteroatoms such as nitrogen, oxygen,sulfur, phosphor, silicon, boron and the like.

The term “amine group” describes an —NR′R″ group as defined hereinabove.Preferably the amine groups in the polyamine structure of the presentinvention are primary amines, where both R′ and R″ are hydrogen, orsecondary amines, where one of R′ and R″ is hydrogen. As is discussedhereinabove, the amine groups of a polyamine structure interact with thecarbonyl portals at the cucurbituril oculi.

The phrase “threading moiety” describes a chemical moiety which iscapable of penetrating into a cucurbituril cavity through thecucurbituril oculi and, preferably, at least a portion thereof iscapable of interacting with the hydrophobic cavity of the cucurbiturilcavitand. Preferred threading moieties according to the presentinvention are therefore hydrophobic chemical moieties, which areterminated or interrupted by the amine groups.

Thus, preferred polyamine structures according to the present inventionare characterized by a chemical structure of which at least a portion ishydrophobic and which can thread into and interact with a cucurbiturilcavity, whereby this portion includes at least two amine groups that caninteract with the polar portals of the cucurbituril.

In order to provide a polyamine structure with an optimal affinitybinding to a cucurbituril or a cucurbituril assembly, the threadingmoiety should be suitably sized to the cucurbituril or to eachcucurbituril in a cucurbituril assembly.

The phrase “suitably sized” with respect to the threading moiety is usedherein to describe a spatial compatibility between the threadingmoieties and the cucurbiturils, which would enable the strongestinteractions between the hydrophobic parts of the threading moiety andthe cucurbituril cavity and between the amino groups and the polarportals of the cucurbituril oculi. Such spatial computability includesboth suitability of the distance between two of the amine groups and thetwo cucurbituril oculi and thus between the length of at least theportion of the threading moiety that include amine groups at both endsand the distance between the polar portals of the cucurbituril oculi,and suitability of a hydrophobic portion of the moiety that lies betweenthe two amine groups and the hydrophobic cavity of a cucurbituril. Forexample, the most suitably sized threading moiety, and also the minimalthreading moiety exhibiting the highest affinity to CB[6], was found tobe an alkyl having 5 to 6 carbon atoms, which terminates with amine atboth ends. Thus, the polyamine structure 1,5-diaminopentane, illustratedin FIG. 3, was found to interact with a CB[6] with the highest observedbinding affinity, K_(D)=10⁻⁶-10⁻⁷ M. Additional description of the sizesuitability, as well as other features that play a significant role inpolyamine-cucurbituril interaction can be found, for example, in Mock,W. L. in Comprehensive Supramolecular Chemistry; Vögtle, F., Ed.;Elsevier Press: New York, 1996; Vol. 2, pp 477-493, which isincorporated by reference as if fully set forth herein.

The exceptional binding affinity between alkylammonium ions and CB[n]swas further found to depend on the degree of fitting between thehydrophobic cavity of the CB[n] and the size and shape of thehydrophobic portion of the threading moiety. The hydrophobic portion ofa PA increases the binding strength between the CB[n] and the PA bydisplacing solvent (water) molecules and creating hydrophobicinteractions in the CB[n] atoms lining the inner surface of the cavity.Thus, the size and shape of the hydrophobic portion affects the bindingaffinity between CB[n] and PA not only due to stearic consideration, butalso in the ability to create favorable surface-to-surface interactioninside the CB[n].

Thus, for example, for cucurbiturils that have a relatively smallcavity, a suitably sized threading moiety would typically be linear(e.g., alkyl, alkenyl, alkynyl, alkoxy, aminoalkyl), whereby forcucurbiturils that have larger cavity, a suitably sized threading moietywould typically be cyclic (e.g., cycloalkyl, aryl and the like).

Thus, depending on the structural features of the cucurbiturils, thethreading moiety according to the present invention can be, for example,an alkyl having 1 to 20 carbon atoms, an alkenyl group having 2 to 20carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an alkoxyhaving 1 to 20 carbon atoms, an aminoalkyl having 1 to 20 carbon atoms,a cycloalkyl having 4 to 20 atoms, a heteroalicyclic having 4 to 7carbon atoms, an aryl having 6 to 20 carbon atoms, a heteroaryl having 5to 20 carbon atoms, as these terms are defined hereinabove, or anycombination thereof.

Preferably the threading moiety is an alkyl having 1 to 10 carbon atoms,an alkenyl group having 2 to 10 carbon atoms, an alkynyl group having 2to 10 carbon atoms, an alkoxy having 1 to 10 carbon atoms, an aminoalkylhaving 1 to 10 carbon atoms, a cycloalkyl having 5 to 10 atoms, aheteroalicyclic having 4 to 10 carbon atoms, an aryl having 6 to 12carbon atoms, a heteroaryl having 6 to 12 carbon atoms or anycombination thereof.

More preferably, the threading moiety is an alkyl having 1 to 6 carbonatoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl grouphaving 2 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, anaminoalkyl having 1 to 6 carbon atoms, a cycloalkyl having 5 to 8 atoms,a heteroalicyclic having 4 to 8 carbon atoms, an aryl having 6 to 12carbon atoms, a heteroaryl having 6 to 12 carbon atoms and anycombination thereof.

Representative examples of threading moieties according to the presentinvention include alkyls having 5 or 6 carbon atoms and those comprisingone or more alkynyl moieties (also referred to herein as polyacetylenicmoieties). The latter are characterized by a rigid structure, whichfacilitates their threading into the cucurbituril cavity and provide asymmetric host-guest structure of the formed affinity pair, as isillustrated in FIGS. 32 a-b.

Since the polyamine structures of the present invention are aimed atbinding to cucurbituril assemblies, which include two or morecucurbituril units, each of the polyamine structures of the presentinvention preferably includes two or more threading moieties, each beinginterrupted or terminating with two amine groups.

The two or more threading moieties are preferably covalently attachedone to another via a branching unit.

As used herein, the phrase “branching unit” describes a chemical moietythat is capable of binding to at least two other chemical moieties,herein the threading moieties, to thereby form a branched structure.

As used herein and is well known in the art, a “branched structure”refers to a non-linear chemical structure.

Representative examples of suitable branching units include, withoutlimitation, amines, branched alkyls, cycloalkyls, heteroalicyclics,branched alkenyls, aryls, heteroaryls, silyls, silicates, boryls,borates, carbamates, thiocarbamates, C-amides, N-amides, S-sulfonamides,N-sulfonamides, ureidos, hydrazines, guanyls and guanidines, as theseterms are defined hereinabove. At least two of the substituents in eachof these units, denoted as R′, R″, R′″ or R* hereinabove, are attachedto at least two threading moieties, either directly or via a spacer, asdescribed hereinabove.

As used herein, the term “silyl” refers to a —SiR′R″— group, wherebyeach of R′, and R″ is as defined hereinabove or, optionally, is athreading moiety.

The term “silicate” refers to a —O—Si(OR′)(OR″)-group, with R′ and R″ asdefined hereinabove.

The term “boryl” refers to a —BR′— group, with R′ as definedhereinabove.

The term “borate” refers to —O—B(OR′)-group, with R′ as definedhereinabove.

The chemical nature of the branching unit, taken together with the sizeand shape of the threading moieties, defines the spatial emplacement ofeach threading moiety in the polyamine structure. This attribute iscrucial for the required fitting between the polyamine structure and thecucurbituril assembly. Therefore, a unique match between a polyaminestructure and the cucurbituril assembly is afforded by a specificspatial emplacement of the CB[n] in the assembly, and the spatialemplacement of the threading moieties in the polyamine structureconcordantly. An exemplary illustration of the above concept ispresented in FIG. 7.

By selecting the appropriate threading moieties and branching units,each of the polyamine structures of the present invention can bedesigned capable of binding to a certain cucurbituril assembly accordingto the present invention, to thereby provide novel PA-CB affinity pairs,such that each affinity pair according to the present inventioncomprises a cucurbituril assembly, as is described hereinabove and apolyamine structure being capable of binding thereto, according to thefeatures outlined hereinabove.

As used herein, the phrase “affinity pair” describes a system thatincludes two molecular structures which are capable of binding one tothe other with a remarkable low dissociation constant, furthercharacterized by high specificity (reciprocities recognition).

As used herein, the term “dissociation constant”, abbreviated by K_(D),represents the equilibrium constant for the decomposition of a complexinto its components in solution, given in molar units M.

An affinity pair according to the present invention can include, forexample, a cucurbituril dimer and a polyamine structure that comprisestwo threading moieties covalently linked therebetween via a branchingunit as is exemplified, for example, in FIGS. 7 and 8. An affinity pairaccording to the present invention can further include atris-cucurbituril structure (a CB trimer) and a polyamine structure thatcomprises three threading moieties, and so on.

Thus, according to the present invention, a plurality of versatileaffinity pairs, each comprised of a certain cucurbituril assembly and asuitable polyamine structure, can be designed and synthesized. Thesingular matching between the polyamine structure and the cucurbiturilassembly of the present invention affords the capacity to designaffinity pairs with such high internal selectivity, which renders suchaffinity pairs highly superior to the presently known affinity pairsystems, and particularly to the well known Av-B system.

A PA-CB affinity pair offers several advantages over the Av-B systemsuch as, for example, controllable and reversible binding afforded bythe versatile variety of PA-CB interactions and the cumulative nature ofthe binding strength, higher specificity stemming from the uniquematching between the polyamine structure and the cucurbituril assembly,low molecular weight, superior chemical stability, wider range ofconjugation chemistry, which is not limited to reactions that are onlycompatible with polypeptides as in the case of the avidin, and noobstruction to spectroscopic measurements aiming at wavelengths relevantto polypeptides.

Following is a broaden description of an exemplary set of advantages ofthe PA-CB affinity pair system over the Av-B affinity pair system.

One highly important advantage of the PA-CB affinity pair according tothe present invention is the fact that the affinity pair can be uniquelydesigned to exhibit any affinity constant, depending on the number andnature of each PA-CB pair in the affinity pair system of the presentinvention. Contrary to that, the Av-B affinity pair is characterized bya fixed dissociation constant of about 10⁻¹⁵ M.

For example, in order to achieve a medium range binding strength, (basedon a K_(D) of about 10⁻⁶ M for each PA-CB pair in the system), anaffinity pair that includes C₅-C₆ alkyl threading moieties in thepolyamine structure and an assembly of cucurbit[6]urils is designed. Forfurther fine-tuning to lower binding strength, polyamine structureincluding shorter or longer threading moieties will afford a suitablebinding constant. For even further fine-tuning, CB[n] of larger internalcavity, such as for example CB[7] and/or CB[8], matched with a suitablealicyclic threading moiety would afford yet more varied bindingconstants.

In order to achieve a high binding strength, in the range of nanomolar,an affinity pair according to the present invention that includes atleast two PA-CB pairs can be designed. For example, in the case of acucurbituril assembly of three CB[6] matched with a polyamine structureof three 5-6 carbon long threading moieties, each suchcucurbituril-polyamine pair contributes approximately 10⁻⁶ M to thebinding strength, amounting to a dissociation constant of 10⁻¹⁸ M of theentire affinity pair system. For further fine-tuning, CB[n] of largerinternal cavity, such as for example CB[7] and/or CB[8], matched with asuitable alicyclic threading moiety affords yet more versatile bindingstrengths. Combined together, the number of PA-CB subpairs in theaffinity pair and the selected polyamine structure and cucurbiturilassembly enable selection of an almost continuous range of bindingstrengths of the affinity pairs of the present invention.

To achieve even higher binding strength, a rigid assembling unit such asa polyaryl or a polyheteroaryl, as defined hereinabove, can be used toconstruct the cucurbituril assembly, as illustrated in FIG. 24. Therigidity of the assembling unit, which provide for the rigidity of thecucurbituril assembly, reduces the free rotations in the assembly andthereby enhances the binding interactions between the assembly and apolyamine structure, resulting in reduced the PA-CB dissociationconstant. An exemplary pathway for synthesis of a rigid cucurbiturilassembly is presented in the Examples section that follows.

In addition, while the Av-B system is susceptible to competitive bindingof other naturally occurring molecules, the PA-CB affinity pair of thepresent invention is characterized by a highly specific recognitionproperty, unique to a given PA-CB construction. It is therefore unlikelythat this unique three-dimensional structure of a given PA-CB affinitypair would be susceptible to any competitive binding of naturallyoccurring biomolecules, and in any unlikely event, a different PA-CBpair can be design so as to overcome these unlikely circumstances.

For example, although naturally occurring polyamines do exist, such asspermine and spermidine, which may bind to e.g., CB[6] with adissociation constant at the micromolar range, such naturally polyamineswill not compete with a polyamine structure, which is specificallydesigned so to match a specific structure of a cucurbituril assembly,and which exhibits a much higher binding constant due to its cumulativeand cooperative binding, for binding to the cucurbituril assembly.

While an Av-B affinity pair exhibits a 56-77 Kilo Dalton molecularweight, mostly due to the avidin part, a factor that weakens theresolution power in many separation analytical techniques, a PA-CBaffinity pair of the present invention is characterized by relativelylower molecular weight. For example, as is demonstrated in FIG. 8, aPA-CB affinity pair exhibiting a binding constant similar to the Av-Bsystem and consisting of a CB[6] dimer having triazine as an assemblingunit, and a matching polyamine structure having two C₅-alkyl threadingmoieties and a tertiary amine as a branching unit, both having pentanoicacid moiety for linking each part of the affinity pair to othermoieties, has a total molecular weight of 2726 grams per mole (2268grams per mole for the cucurbituril assembly and 458 grams per mole forthe polyamine structure). In sharp contrast, the Av-B affinity pairexhibits a 58000 to 76000 gram per mole molecular weight.

While avidin is a protein, being susceptible to temperature-, pH-, andchemical environment-related denaturation, and limiting the chemistry bywhich it can be conjugated to relevant moieties, a PA-CB pair is stableand durable, and is thus a much more robust affinity pair as comparedwith Av-B. The chemistry that can be applied when conjugating desiredmoieties to a PA-CB affinity pair is therefore almost unlimited.

Furthermore, while avidin is known to obscure specrtophotometric signalsrelevant to polypeptides, a PA-CB affinity pair, being devoid of apolypeptide chain, will remain transparent at these wavelengths.

The affinity pairs of the present invention can therefore be utilized invarious applications, as in the case of an Av-B system, when one or morefunctional moieties are attached to the cucurbituril assembly and/or tothe polyamine structure.

Hence, according to preferred embodiments of the present invention, eachof the cucurbituril assemblies herein described and each of thepolyamine structures, either alone or when forming an affinity pair, canfurther comprise one or more functional moieties attached thereto.

As used herein, the phrase “functional moiety” refers to a molecule or aplurality of molecules characterized by one or more functionalities thatcan be used in a particular application, as is exemplified hereinbelow.

Each functional moiety according to the present invention can beattached to the cucurbituril assembly and/or the polyamine structure ofthe present invention by any acceptable means such as, for example,covalently, electrostatically, and the like.

The functional moiety can be attached to different components of thecucurbituril assembly and/or the polyamine structure of the presentinvention, as is detailed hereinunder.

In one example, a functional moiety can be attached to a derivatizedcucurbituril unit in a cucurbituril assembly via a functional grouppresent in the derivatized cucurbituril. As is exemplified in FIG. 6, aCB[6] can be attached to a DNA oligomer via a secondary amine functionalgroup by, for example, reacting the 5′ alkyl phosphate group of the DNAoligomer with carbodiimide, which forms a phosphate ester, andsubsequently coupling the ester to a cucurbituril derivatized by anamine group, so as to form a stable phosphoramidate linkage.

In another example, a functional moiety can be attached to an assemblingunit in a cucurbituril assembly, as is exemplified in FIG. 7 (Compound24).

In yet another example, a functional moiety can be attached to athreading moiety of a polyamine structure, as is exemplified in FIG. 3(Compound 7). Alternatively, a functional moiety can be attached to abranching unit in the polyamine structure as is exemplified in FIG. 7(Compound 23).

While PA-CB affinity pairs have been practiced in some applications,such affinity pairs that further comprise one or more functionalmoieties attached to a cucurbituril or a polyamine have never beensuggested, prepared or utilized heretofore. The present inventiontherefore further encompasses affinity pairs that comprise a singlecucurbituril, optionally derivatized, and a suitable polyamine boundthereto, whereby one or more functional moieties, as described herein,are attached to the cucurbituril and/or the polyamine structure.

Representative examples functional moieties that when attached to acucurbituril and/or a polyamine structure according to the presentinvention can be efficiently utilized in various applications include,without limitations, pharmaceutically active agents, biomolecules, andlabeling moieties.

As used herein, the phrase “pharmaceutically active agent” describes amolecule or a plurality of molecules that exert one or morepharmaceutical activities.

Representative examples of pharmaceutically active agents that areusable in the context of the present invention include, withoutlimitation, anti-proliferative agents, anti-inflammatory agents,antibiotics, anti-viral agents, anti-oxidants, anti-hypertensive agents,chemosensitizing agents, ligands, inhibitors, agonists, antagonists,hormones, vitamins and co-factors.

Non-limiting examples of chemotherapeutic agents include aminocontaining chemotherapeutic agents such as daunorubicin, doxorubicin,N-(5,5-diacetoxypentyl)doxorubicin, anthracycline, mitomycin C,mitomycin A, 9-amino camptothecin, aminopertin, antinomycin, N⁸-acetylspermidine, 1-(2-chloroethyl)-1,2-dimethanesulfonyl hydrazine,bleomycin, tallysomucin, and derivatives thereof; hydroxy containingchemotherapeutic agents such as etoposide, camptothecin, irinotecaan,topotecan, 9-amino camptothecin, paclitaxel, docetaxel, esperamycin,1,8-dihydroxy-bicyclo[7.3.1]trideca-4-ene-2,6-diyne-13-one, anguidine,morpholino-doxorubicin, vincristine and vinblastine, and derivativesthereof, sulfhydril containing chemotherapeutic agents and carboxylcontaining chemotherapeutic agents.

Non-limiting examples of antibiotics include benzoyl peroxide,octopirox, erythromycin, zinc, tetracyclin, triclosan, azelaic acid andits derivatives, phenoxy ethanol and phenoxy proponol, ethylacetate,clindamycin and meclocycline; sebostats such as flavinoids; alpha andbeta hydroxy acids; and bile salts such as scymnol sulfate and itsderivatives, deoxycholate and cholate.

Non-limiting examples of non-steroidal anti-inflammatory agents includeoxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam, andCP-14,304; salicylates, such as aspirin, disalcid, benorylate,trilisate, safapryn, solprin, diflunisal, and fendosal; acetic acidderivatives, such as diclofenac, fenclofenac, indomethacin, sulindac,tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin,fentiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac;fenamates, such as mefenamic, meclofenamic, flufenamic, niflumic, andtolfenamic acids; propionic acid derivatives, such as ibuprofen,naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen,indopropfen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen,tioxaprofen, suprofen, alminoprofen, and tiaprofenic; pyrazoles, such asphenylbutazone, oxyphenbutazone, feprazone, azapropazone, andtrimethazone.

Non-limiting examples of steroidal anti-inflammatory drugs include,without limitation, corticosteroids such as hydrocortisone,hydroxyltriamcinolone, alpha-methyl dexamethasone,dexamethasone-phosphate, beclomethasone dipropionates, clobetasolvalerate, desonide, desoxymethasone, desoxycorticosterone acetate,dexamethasone, dichlorisone, diflorasone diacetate, diflucortolonevalerate, fluadrenolone, fluclorolone acetonide, fludrocortisone,flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortinebutylesters, fluocortolone, fluprednidene(fluprednylidene)acetate,flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisonebutyrate, methylprednisolone, triamcinolone acetonide, cortisone,cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate,fluradrenolone, fludrocortisone, diflurosone diacetate, fluradrenoloneacetonide, medrysone, amcinafel, amcinafide, betamethasone and thebalance of its esters, chloroprednisone, chlorprednisone acetate,clocortelone, clescinolone, dichlorisone, diflurprednate, flucloronide,flunisolide, fluoromethalone, fluperolone, fluprednisolone,hydrocortisone valerate, hydrocortisone cyclopentylpropionate,hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone,beclomethasone dipropionate, triamcinolone, and mixtures thereof.

Non-limiting examples of anti-oxidants include ascorbic acid (vitamin C)and its salts, ascorbyl esters of fatty acids, ascorbic acid derivatives(e.g., magnesium ascorbyl phosphate, sodium ascorbyl phosphate, ascorbylsorbate), tocopherol (vitamin E), tocopherol sorbate, tocopherolacetate, other esters of tocopherol, butylated hydroxy benzoic acids andtheir salts, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid(commercially available under the trade name Trolox®), gallic acid andits alkyl esters, especially propyl gallate, uric acid and its salts andalkyl esters, sorbic acid and its salts, lipoic acid, amines (e.g.,N,N-diethylhydroxylamine, amino-guanidine), sulfhydryl compounds (e.g.,glutathione), dihydroxy fumaric acid and its salts, lycine pidolate,arginine pilolate, nordihydroguaiaretic acid, bioflavonoids, curcumin,lysine, methionine, proline, superoxide dismutase, silymarin, teaextracts, grape skin/seed extracts, melanin, and rosemary extracts.

Non-limiting examples of vitamins include vitamin A and its analogs andderivatives: retinol, retinal, retinyl palmitate, retinoic acid,tretinoin, iso-tretinoin (known collectively as retinoids), vitamin E(tocopherol and its derivatives), vitamin C (L-ascorbic acid and itsesters and other derivatives), vitamin B₃ (niacinamide and itsderivatives), alpha hydroxy acids (such as glycolic acid, lactic acid,tartaric acid, malic acid, citric acid, etc.) and beta hydroxy acids(such as salicylic acid and the like).

Non-limiting examples of hormones include androgenic compounds andprogestin compounds such as methyltestosterone, androsterone,androsterone acetate, androsterone propionate, androsterone benzoate,androsteronediol, androsteronediol-3-acetate,androsteronediol-17-acetate, androsteronediol 3-17-diacetate,androsteronediol-17-benzoate, androsteronedione, androstenedione,androstenediol, dehydroepiandrosterone, sodium dehydroepiandrosteronesulfate, dromostanolone, dromostanolone propionate, ethylestrenol,fluoxymesterone, nandrolone phenpropionate, nandrolone decanoate,nandrolone furylpropionate, nandrolone cyclohexane-propionate,nandrolone benzoate, nandrolone cyclohexanecarboxylate,androsteronediol-3-acetate-1-7-benzoate, oxandrolone, oxymetholone,stanozolol, testosterone, testosterone decanoate, 4-dihydrotestosterone,5α-dihydrotestosterone, testolactone, 17α-methyl-19-nortestosterone andpharmaceutically acceptable esters and salts thereof, and combinationsof any of the foregoing, desogestrel, dydrogesterone, ethynodioldiacetate, medroxyprogesterone, levonorgestrel, medroxyprogesteroneacetate, hydroxyprogesterone caproate, norethindrone, norethindroneacetate, norethynodrel, allylestrenol, 19-nortestosterone, lynoestrenol,quingestanol acetate, medrogestone, norgestrienone, dimethisterone,ethisterone, cyproterone acetate, chlormadinone acetate, megestrolacetate, norgestimate, norgestrel, desogrestrel, trimegestone,gestodene, nomegestrol acetate, progesterone, 5α-pregnan-3β,20α-diolsulfate, 5α-pregnan-3β,20β-diol sulfate, 5α-pregnan-3β-ol-20-one,16,5α-pregnen-3β-ol-20-one, 4-pregnen-20β-ol-3-one-20-sulfate,acetoxypregnenolone, anagestone acetate, cyproterone, dihydrogesterone,flurogestone acetate, gestadene, hydroxyprogesterone acetate,hydroxymethylprogesterone, hydroxymethyl progesterone acetate,3-ketodesogestrel, megestrol, melengestrol acetate, norethisterone andmixtures thereof.

Chemosensitizing agents, ligands, inhibitors, agonists, antagonists, andco-factors can be selected according to a specific indication.

Affinity pairs having one or more pharmaceutically active agentsattached thereto, optionally in combination with another functionalmoiety, can be used, for example, for drug delivery and bioactivityscreening.

As used herein, the phrase “labeling moiety” refers to a detectablemoiety or a probe and includes, for example, chromophores, fluorescentcompounds, phosphorescent compounds, heavy metal clusters, andradioactive labeling compounds, as well as any other known detectablemoieties.

As used herein, the term “chromophore” refers to a chemical moiety that,when attached to another molecule, renders the latter colored and thusvisible when various spectrophotometric measurements are applied.

The phrase “fluorescent compound” refers to a compound that emits lightat a specific wavelength during exposure to radiation from an externalsource.

The phrase “phosphorescent compound” refers to a compound emitting lightwithout appreciable heat or external excitation as by slow oxidation ofphosphorous.

A heavy metal cluster can be for example a cluster of gold atoms used,for example, for labeling in electron microscopy techniques.

As used herein the term “biomolecules” refers to a naturally-occurringmolecule, a synthetic analog or a modification thereof, which ischaracterized by a biological functionality and include, for example, anamino acid, a peptide, a protein, an antibody, an antigen, a nucleicacid, a polynucleotide, an oligonucleotide, an antisense, apolysaccharide, a fatty acid, a membrane and a cell.

Alternatively, the functional moiety may form a part of a solid support,such that at least one component of the affinity pair, namely, thepolyamine or a cucurbituril, are attached to a solid support.

The phrase “solid support” as used herein encompasses solid supportssuch as, but not limited to, solid surfaces of any material, polymers,resins, beads, including plastic beads, metal beads and magnetic beads,silica matrices such as glass and sol-gel particles and the like.

The term “surface” as used herein refers to any outer boundary of anartifact or a material layer constituting or resembling such a boundary.

The term “polymer” refers to a naturally occurring or synthetic compoundconsisting of large molecules made up of a linked series of repeatedsimple monomers.

The term “resin” refers to any of a class of solid or semisolid viscoussubstances obtained either as exudations from certain plants or preparedby polymerization and cross-linking of simple molecules.

The phrase “sol-gel” as used herein refers to a versatile solutionprocess for making ceramic and glass materials. In general, the sol-gelprocess involves the transition of a system from a liquid “sol” (mostlycolloidal) into a solid “gel” phase. Applying the sol-gel process, it ispossible to fabricate ceramic or glass materials in a wide variety offorms: ultra-fine or spherical shaped powders, thin film coatings,ceramic fibers, microporous inorganic membranes, monolithic ceramics andglasses, or extremely porous aerogel materials.

The high, effective and advantageous affinity between the cucurbiturilassemblies and the polyamine structures described above can be utilizedin various affinity binding methods.

Such methods are performed by contacting a cucurbituril or cucurbiturilassembly with a suitable polyamine structure, designed as described indetail hereinabove, to thereby provide the affinity pair, preferablyunder conditions that enable protonation of the polyamine structure,e.g., acidic conditions.

Protonation of the polyamine structure enhance the binding interactionsbetween the carbonyl groups at the cucurbituril portals and the aminegroups of the polyamine structures.

Whenever one or more functional moieties, as described above, areattached to the thus formed affinity pair, the affinity binding methodsof the present invention can be used in a vast variety of applications.

Some non-limiting examples for the diverse embodiments of the methods ofaffinity binding according to the present invention include, but are notlimited to, isolation and purification of biological molecules viaaffinity chromatography, immunohistochemical staining, introducingmultiple labels into tissues, localizing hormone binding sites, flowcytometry, in situ localization and hybridization, radio-, enzyme-, andfluorescent immunoassays, neuronal tracing, genetic mapping, hybridomascreening, purification of cell surface antigens, coupling of antibodiesand antigens to solid supports, examination of membrane vesicleorientation, and drug delivery.

Thus, in one exemplary embodiment of the methods of the presentinvention, the affinity binding of a cucurbituril or a cucurbiturilassembly and a polyamine structure according to the present invention isused in immunohistochemical staining. As is well known in the art, a keyto successful identification of proteins in tissues and other samples byimmunohistochemical staining involves careful selection of theprotein-specific antibody and an efficient affinity pair to conjugatethe antibody to a chromogen, a compound that can be converted to apigment. An immunohistochemical staining, according to this embodiment,can be performed, for example, by attaching a plurality of the polyaminestructures of the present invention to the antibody; attaching achromogen such as hematoxylin to a cucurbituril assembly, whereby thepolyamine structures and the cucurbituril assembly are selected so as toefficiently affinity bind one to another, and applying the method of thepresent invention to thereby provide an affinity pair attached to anantibody at one end and to the chromogen at the other end.

In another exemplary embodiment of the methods of the present invention,the affinity binding of a cucurbituril or a cucurbituril assembly and apolyamine structure according to the present invention is used inimmunoassays. The immunoassays can be homogeneous or heterogeneous andinclude, for example, the EMIT® assay described in U.S. Pat. No.3,817,837, the CEDIA assay, the radioimmunoassay (RIA),immunofluorescence methods, enzyme-linked immunoassays, such as theenzyme-linked immunosorbent assay (ELISA) and so forth.

In another exemplary embodiment of the methods of the present invention,the affinity binding of a cucurbituril or a cucurbituril assembly and apolyamine structure according to the present invention is used in flowcytometry. As a well established technique, flow cytometry involves theuse of a beam of laser light projected through a liquid stream thatcontains cells, or other particles, which when subjected to the focusedlight emit detectable signals. These signals are then converted forcomputer storage and data analysis, and can provide information aboutvarious cellular properties. In order to make the measurement ofbiophysical or biochemical properties of interest possible, the cellsare usually stained with fluorescent dyes that bind specifically tospecific cellular constituents. The dyes are excited by the laser beam,and emit light at a different wavelength. A flow cytometry experiment,according to this embodiment, can be performed, for example, byattaching a plurality of the polyamine structures to specific cellularconstituents; attaching a fluorescent dye, such as fluorescein orresorcinolphthalein, to a cucurbituril assembly, whereby the polyaminestructures and the cucurbituril assembly are selected so as toefficiently affinity bind one to another, and applying the method of thepresent invention to thereby provide an affinity pair attached to acertain type of cells at one end and to the fluorescent dye at the otherend.

In yet another exemplary embodiment of the methods of the presentinvention, the affinity binding of a cucurbituril or a cucurbiturilassembly and a polyamine structure according to the present invention isused in fluorescence in situ hybridization (FISH). FISH is a method oflocalizing, either mRNA within the cytoplasm or DNA within thechromosomes of the nucleus, by hybridizing the sequence of interest to acomplimentary strand of a nucleotide probe labeled with a fluorescentdye. The method is also called chromosome painting. The sensitivity ofthe technique is such that threshold levels of detection are in theregion of 10-20 copies of mRNA or DNA per cell. Probes are complimentarysequences of nucleotide bases to the specific RNA or DNA sequence ofinterested. These probes can be as small as 20-40 base pairs, up to a1000 base pairs. Types of probes can be oligonucleotide, single ordouble stranded DNA and RNA strands which are labeled with a fluorescentdye. A FISH procedure, according to this embodiment, can be performed,for example, by attaching a polyamine structure of the present inventionto a nucleotide probe; attaching a fluorescent dye, such as fluoresceinor resorcinolphthalein, to a cucurbituril assembly, whereby thepolyamine structures and the cucurbituril assembly are selected so as toefficiently affinity bind one to another, and applying the method of thepresent invention to thereby provide an affinity pair attached to anucleotide probe at one end and to the fluorescent dye at the other end.

In another exemplary embodiment of this aspect of the present invention,at least one of the functional moieties forms a part of a solid supportand the method is used for affinity chromatography.

Affinity chromatography is a well known technology that uses aninsoluble substance exhibiting a selective capacity to bind a particularchemical or biochemical entity from a mixture of compounds, e.g., insolution. Description of techniques of affinity chromatography used forseparation of biochemical entities are described for example in “Methodsin Enzymology”, vol. 34, edited by W. B. Jakoby and M. Wilcheck,Academic Press (1975). These techniques may be practiced by attaching apolyamine structure or a cucurbituril or cucurbituril assembly to asolid support typically used for chromatography, modified so as to havefunctional moieties that can be attached to the affinity pair componentor to other functional moieties attached thereto, and eluting a solutionto be separated in which the desired biological entity is attached tothe complementary component of the affinity pair. The desired compoundis thus isolated from the mixture and can be thereafter released bydetachment of the affinity pair.

Such techniques can be efficiently utilized using the affinity pairs ofthe present invention particularly since the affinity binding betweenthe cucurbiturils and polyamines is pH- and solvent-dependent. Thus, bymodifying the elution conditions (e.g., the solvents), binding andreleasing of the desired compounds can be easily performed.

The same technique can be utilized, for example, for isolating onecomponent of the affinity pairs of the present invention, by attachingthe complementary components to a chromatography solid support. Bydesigning polyamine structures or cucurbiturils that can specificallyinteract one with the other, specific polyamine structures or specificcucurbiturils or cucurbiturils assembly can be isolated from mixturescontaining a plurality of similar yet distinct components.

In one exemplary embodiment, such a method can be utilized for isolatinga cucurbituril from a mixture containing same, which is effected byproviding a column packed with a polyamine structure designed capable ofselectively binding to the desired cucurbituril, eluting a mixturecontaining the desired cucurbituril through the column, to therebyobtain a column having at least a portion of the desired cucurbiturilbound thereto and thereafter releasing the desired cucurbituril from thecolumn.

As is described in detail in the Examples section that follows, such amethod can be efficiently utilized for isolating rare cucurbiturils andthus for enriching a cucurbituril reaction mixture for rarecucurbiturils. As is discussed hereinabove, the isolation of pure CB[n]sand particularly of pure rare CB[n]s has become the major impediment totheir availability, particularly when large-scale synthesis is required,a limitation that is solved using the technique described herein.

In addition to the diagnostic, analytical and therapeutic applicationsdescribed above, a myriad of applications can benefit from having theability of binding two or more entities in order to create newfunctions. For example, by binding one or more electrochemically activeor photochemically active groups to a biomolecule, one can create newphenomena, such as electric conductivity, photoconductivity, uniquephotochemical phenomena, and the like.

Another benefit could originate from the capacity to attach anelectrostatically charged or a magnetic moiety to a molecule. Theseconjugated molecules may be spatially ordered in either electric ormagnetic fields in solution or in low-dimensional environments so as toform ordered structures either in two or three dimensions, such as inmonolayers (either physically adsorbed on surface or chemically linkedto the surface), liquid crystals (either thermotropic or lyotropic) andthree-dimensional single crystals.

In general, the affinity binding methods according to the presentinvention can be used in Diagnostics and analytical applications,wherein the functional moiety is typically a detectable moiety;Therapeutics (as prodrugs, targeting structures, conjugates, and forslow release applications); for employment of new physical properties;for creating new chemical reactivities; and for forming new molecularand supramolecular structures.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate the invention in a non limiting fashion.

Example 1 Design and Preparation of an Affinity ChromatographyPurification Strategy

As is detailed hereinabove, the strong binding interactions betweenCB[n]s and protonated amines or diamines have been harnessed fordesigning an affinity chromatography purification strategy. To that end,polymer-bound polyamines were prepared and employed as follows, takingadvantage of the fact that their binding affinities to cucurbiturils aresolvent- and pH-dependent.

Preparation of Highly-Dense Chloromethylated Polystyrene Beads

Chloromethylated polystyrene beads (Merrifield resins) having a higherdensity of functional groups, as compared with commercially availableresins, were prepared as follows:

A resin (8 grams) of cross-linked polystyrene beads (Rohm & Haas,amberlite XE-305, 2% divinylbenzene, 20-50 mesh, average pore size 1400angstroms, surface area 48 m²/gram), was mixed with chloromethylmethylether (75 ml), tetrachlorostannane (2.4 ml, prepared according to Trostand Keinan, J. Am. Chem. Soc. 1978, 100, 7779; and Pepper et al. J.Chem. Soc. 1953, 4097) was added dropwise, and the resulting mixture wasrefluxed for 3 hours. The mixture was then cooled to room temperature,poured into methanol (200 ml) and filtered. The obtained beads werewashed thoroughly with methanol and were thereafter dried under reducedpressure to give a highly dense chloromethylated resin (FIG. 3, Compound5).

Elemental analysis of the obtained chloromethylated resin (C, 71.38; H,5.99; Cl, 19.73.) indicated loading of 2.7 mmol/grams (85% ringsubstitution).

The IR spectrum of the chloromethylated resin, presented in FIG. 10,showed characteristic peaks at 1611, 1510, 1442, 1420 cm⁻¹.

Preparation of Polymer-Bound Polyamines (According to Manov, N.; Bienz,S. Tetrahedron 2001, 57, 7893)

The chloromethylated resin obtained as described above (FIG. 3, Compound5, 7 grams) was mixed with DMF (50 ml), followed by dropwise addition ofa solution of the tosylate salt of monotert-butoxycarbonyl-1,5-diaminopentane (Novabiochem, 5.2 grams, 14 mmol)in a pyridine-DMF solution (2:1, 30 ml). The resulting mixture wasagitated at 50° C. for 24 hours, and was thereafter filtered, washedconsecutively with DMF, CH₂Cl₂ and MeOH and dried under reducedpressure, to give a Boc derivative of an aminated resin (FIG. 3,Compound 6).

Elemental analysis of the obtained aminated resin (C, 64.06; H, 6.61; N,4.65.) indicated loading of 1.67 mmol/gram.

The IR spectrum of the Boc protected aminated resin, presented in FIG.11, showed characteristic peaks at 3381, 1631, 1485, 1450, 1425 cm⁻¹.

Removal of the Boc protecting group was performed by packing theBoc-protected aminated resin (3 grams) in a 1×12 cm column and washingit with trifluoroacetic acid (10% v/v in CH₂Cl₂, 50 ml) at a flow rateof 1 ml/minute. The column was then washed with CH₂Cl₂ (50 ml) andfinally with DMF (50 ml) at a flow rate of 2 ml/minute. The IR spectrumof the resulting resin, presented in FIG. 12, indicated that a fullyprotonated aminated resin (FIG. 3, Compound 7) was obtained.

Example 2 Affinity Purification of Dimethylcyclopentano-CB

Dimethylcyclopentano-CB (FIG. 4, Compound 3) was synthesized accordingto the procedures described by Day et al. (J. Org. Chem. 2001, 66, 8094)and Isobe et al. (Org. Lett. 2002, 4, 1287), by heating at 90° C. for 24hours, a 5:1 mixture of glycoluril (Compound 2) anddimethylcyclopentano-glycoluril (FIG. 4, Compound 3), and formaldehydein the presence of concentrated sulfuric acid (FIG. 4), so as to givethe desired cucurbituril product in 30% yield. The water-solublefraction (740 mg) of the crude, heterogeneous mixture (1.3 gram) wasdissolved in neutral water (50 ml) and passed through a column loadedwith the protonated aminated resin prepared as described above, at aflow rate of 0.5 ml/minute (of the heterogeneous mixture in water). Thecolumn was then washed sequentially with water, methanol, CH₂Cl₂, andagain with methanol. Removal of the solvent from the combined eluentafforded a solid residue (510 mg). This residue was used for collectinga second harvest of CB[6] using the same column, as is detailedhereinbelow.

A sample of the resin was dried under reduce pressure overnight and wasanalyzed by FTIR. The obtained spectrum, presented in FIG. 13, indicatedthat the resin is indeed loaded with the CB[6].

The resin-bound CB[6] was released from the column by elution with a 1:2mixture of triethylamine-DMF (100 ml) at a flow rate of 2 ml/minute. Thecolumn was thereafter washed with water (200 ml), and the combinedeluent was concentrated under reduced pressure. Methanol was then addedand the precipitate was collected and dried. The resultant white powder(195 mg) was analyzed by ¹H NMR and ESI-MS and the analyses arepresented in FIGS. 14 and 15, respectively. As is shown in FIGS. 14 and15, the resin-bound CB[6], obtained as a powder upon elution from thecolumn, contained mainly a mixture of Compound 4 (FIG. 4) and Compound 1(FIG. 2), with a small amount of di(dimethylcyclopentano)-CB[6].

Regeneration of the column was performed by washing it with 10% (v/v)trifluoroacetic acid in CH₂Cl₂. The regenerated column was used again toharvest additional amounts of CB[6] from the CB-depleted remnantsobtained in the first harvest (510 mg). That residue was dissolved inneutral water and loaded on the column as described above. Unloading thecolumn with triethylamine-DMF yielded a second crop of pure CB[6] (165mg).

The performance of the column over multiple cycles of affinitychromatography was evaluated by loading and unloading a sample ofpurified CB[6] (150 mg) four times. The sample was trapped and releasedquantitatively in all eight operations with no apparent loss of thecolumn capacity.

Example 3 Affinity Separation of a Thermodynamically Unfavorable CB

The use of the above described affinity chromatography technique forisolating rare CB derivatives, which are highly beneficial in variousapplications, as is detailed hereinabove, was evaluated. The rarecucurbituril derivative hexacyclohexano-CB[6] was chosen as a firstrepresentative example.

Thus, a mixture of pentacyclohexano-CB[5] (FIG. 5, Compound 10) andhexacyclohexano-CB[6] (FIG. 5, Compound 11) was prepared by reactingcyclohexanoglycoluril (FIG. 5, Compound 9) with formaldehyde, accordingto the procedure described by Zhao, J et al. (Angew. Chem. Int. Ed.2001, 40, 4233). Compound 10 and Compound 11 were obtained in 16% and 2%yield, respectively.

Using the affinity chromatography described above, the minor CB[6]product, Compound 11, was rapidly separated from the crude reactionmixture. The NMR spectra of the mixture before (data not shown) andafter (FIG. 16) the separation indicated that pure Compound 11 wasquantitatively recovered from the product mixture. The purity ofCompound 11 was evident from its ¹H-NMR (FIG. 16), ¹³C-NMR (FIG. 17) andMS data (FIG. 18).

Example 4 Separation of the Rare CB Dodecamethycucurbit[6]Uril

Another rare cucurbituril derivative, decamethylcucurbit[6]uril (FIG. 5,Compound 14) was chosen for further demonstrating the utility of theabove described affinity chromatography technique.

Flinn et al. (Angew. Chem. Int. Ed. Engl., 1992, 31, 1475-1477) reportedthat in the reaction of dimethylglycoluril (FIG. 5, Compound 12), theCB[5] product is obtained in a higher selectivity as compared withcyclohexanoglycoluril, and that only one product,decamethylcucurbit[5]uril (FIG. 5, Compound 13), could be isolated fromthe reaction mixture.

A mixture of decamethylcucurbit[5]uril (Compound 13) anddodecamethycucurbit[6]uril (Compound 14) was prepared by reactingdimethylglycoluril (Compound 12) with formaldehyde, according to theprocedure described by Flinn et al. (supra).

Using the affinity chromatography described above, the minor CB[6]product, Compound 14, was separated from the crude reaction mixture, andwas obtained in 0.2% yield, thus demonstrating the efficacy of theaffinity chromatography of the present invention The purity of Compound14 was evident from its ¹H-NMR (FIG. 19), ¹³C-NMR (FIG. 20) and MS data(FIG. 21).

Example 5 General Procedure for the Preparation of a CucurbiturilAssembly

A CB[n]s assembly according to the present invention can be prepared viatwo major synthetic pathways: one pathway involves formation of aderivatized CB monomer prior to the assembling step and the otherinvolves formation of an assembling unit to which several derivatizedglycoluril moieties are attached and are thereafter converted into CBunits on the assembly unit.

According to the first synthetic pathway, a derivatized CB monomer,which includes a functional group that can be thereafter attached to anassembling unit or utilized to form an assembling unit, is prepared, forexample, by reacting a mixture of a glycoluril and a derivatizedglycoluril and formaldehyde or any other aldehyde, under acidicconditions. The derivatized cucurbituril is preferably isolated, as ispresented and exemplified hereinabove, using the affinity chromatographymethod of the present invention.

An assembly of two or more such derivatized CBs attached to anassembling unit is then formed, using, depending on the nature of thefunctional group and the assembling unit, any suitable technique.Alternatively, the functional group on the derivatized CB is eitherdirectly reacted or is converted to another functional group, which canbe reacted so as to form a ring or any other conjugate with suitablefunctional groups of other derivatized CB(s), to thereby form thecucurbituril assembly. For example, in case where the functional groupis amine and the assembling unit is triazine, derivatized cucurbiturilsattached to the assembling unit are prepared by a cyanoamine cyclizationreaction.

According to the second pathway, a derivatized glycoluril having afunctional group is first prepared. Two or more of such derivatizedglycolurils attached to an assembling unit are then prepared, using,depending on the nature of the functional group and the assembling unit,any suitable technique. Alternatively, the functional group on thederivatized glycoluril is either directly reacted or is converted toanother functional group, which can be reacted so as to form a ring orany other conjugate with suitable functional groups of other derivatizedglycoluril(s). For example, in case where the functional group is amineand the assembling unit is triazine, derivatized glycolurils attached tothe assembling unit are prepared by a cyanoamine cyclization reaction.

The thus formed compound is then reacted with other derivatized ornon-derivatized glycolurils, in the presence of formaldehyde, at apre-selected ratio, to thereby form a cucurbituril assembly in whicheach cucurbituril is attached to the assembling unit.

In each of the pathways described above, a functional moiety such as,for example, a pharmaceutically active agent, a labeling compound, abiomolecule, or a solid support can be attached, at any stage, either tothe derivatized cucurbituril, the derivatized glycoluril, or to theassembling unit, to thereby produce a cucurbituril assembly having afunctional moiety attached thereto.

For example, in the first pathway, a derivatized cucurbituril having afunctional group for forming an assembly and another functional groupfor attaching a functional moiety can be prepared. During the attachmentto the assembling unit the second functional group is protected and oncethe cucurbituril assembly is formed, this functional group isdeprotected and reacted with a functional moiety, using knowntechniques.

In another example, in the second synthetic pathway, the derivatizedglycoluril units that are attached to the assembling units are reactedwith glycolurils that have a protected functional group. Once thecucurbituril assembly is formed, the functional group is deprotected andreacted with the functional moiety using known techniques.

Example 6 Preparation of a Cucurbituril Trimer

A CB trimer, according to the present invention, can be preparedaccording to two converging pathways, as is detailed hereinabove(Example 5) and is further presented in FIG. 6. In general, one route ofsynthesizing a CB trimer involves formation of a derivatized CB monomerprior to the trimerization step, and another route involves formation ofan assembling unit on which three CB moieties are formed in aconsecutive step.

As an exemplary starting material for both routes, a glycolurilderivatized by a fused pyrrolidine ring (FIG. 6, Compound 19) isprepared, either by reacting diethyl iminodiacetate (FIG. 6, Compound15) with 4-morpholinyl-furan-3-one (FIG. 6, Compound 16), or by reactingdimethylester-glycoluril (FIG. 6, Compound 17) and2,5-dione-pyrollidono-glycoluril (FIG. 6, Compound 18).

Compound 19 is reacted with glycoluril, Compound 2, in a 1:5 ratio withformaldehyde, in the presence of concentrated sulfuric acid, to therebyform the derivatized cucurbituril pyrrolidino-CB[6] (FIG. 6, Compound20). Compound 20 is thereafter converted to the cyanoamine and thelatter is reacted so as to form a CB trimer (as presented in FIG. 6,Compound 22) having a triazine ring as the assembling unit.

Alternatively, Compound 19 is first converted to the cyanoimine, whichis thereafter reacted so as to form the triazine ring at the center oftrimerization. The resulting1,3,5-tri(2,5-dione-pyrollidono-glycoluril)-triazine (FIG. 6, Compound21) is reacted with glycoluril, Compound 2, in a 1:15 ratio and withformaldehyde, in the presence of concentrated sulfiric acid, to therebyform a cucurbituril trimer (FIG. 6, Compound 22) having a triazine ringas the assembling unit.

Example 7 Preparation of Derivatized Cucurbiturils, a CucurbiturilTrimer and a CB-Protein Conjugate

Due to the non-trivial preparation of derivatized CBs, only very fewsuch compounds have been prepared to date.

While the present invention is aimed, inter alia, at systematicallygenerating derivatized cucurbiturils, for the purpose of formingcucurbituril assemblies and/or attachment of various functional moietiesthereto, a general pathway for generating an exemplary derivatizedcucurbituril, CB[5,1] has been developed, as is presented in FIGS. 22and 23 (Compounds 35 and 36). As is further exemplified in FIG. 23, suchderivatized cucurbiturils can be attached to a variety of functionalmoieties, as defined hereinabove, for example a protein (FIG. 23,Compound 37), or to a cucurbituril assembly such as presented in FIG. 23as Compound 38.

Hence, a number of substituted phenyl glycoluril (FIG. 23, Compound 35),have prepared from the corresponding substituted benzil derivatives,(FIG. 23, Compound 34), as follows:

Preparation of Ditolyl Glycoluril (FIG. 22, Compound 26)

Trifluoroacetic acid (TFA, 6 ml) was added to a solution of urea (6.0grams, 0.1 moles) and tolylbenzil (Compound 25, 11.90 grams, 0.05 moles)in benzene (200 ml), and the resulting mixture was refluxed for 12hours, using a Dean-Stark trap, until water distillation was no longerobserved. The resulting white solid was then filtered, washed with coldethanol and dried under high vacuum, to give 21.5 grams (73%) of theproduct. The structure and purity of Compound 26 were evident from its¹H NMR spectrum (300 MHz; DMSO), which showed a methyl at 2.35 ppm(singlet, 6H); an aromatic ring at 7.01 ppm (multiplet, 10H); and aminehydrogen at 7.90 ppm (singlet, 4H).

Preparation of Para-Dicarboxylic Diphenyl Glycoluril (FIG. 22, Compound27)

The above glycoluril was synthesized based on Elemans et al. (J. Org.Chem. 2003, 68, 9040-9049). In brief, a suspension of Compound 26 (2.0grams, 8.4 mmoles) and KMnO₄ (6.66 grams, 42 mmoles) in water (100 ml)was refluxed for 16 hours. The resulting brown suspension was cooled andfiltered over celite and the residue was washed with 50 ml of aqueous 1N NaOH. The pale yellowish filtrate was acidified to pH 1 with aqueous37% HCl while stirring vigorously. The precipitate was filtered, washedwith 200 ml of water, and dried under vacuum, to yield 1.8 grams (80%)of Compound 27 as a white powder. The structure and purity of Compound27 were confirmed by its ¹H NMR spectrum:

¹H NMR (300 MHz; DMSO): δ=7.85 ppm (doublet, 4H, 3J, 8.2 Hz), 7.30 ppm(doublet, 4H, 3J 8.2 Hz) and 7.9 ppm (broad peak, 4H).

Preparation of Para-Monocarboxylic Diphenyl Glycoluril (FIG. 22,Compound 30)

A suspension of Compound 29 (1.0 grams, 4 mmoles) and KMnO₄ (2 grams, 13mmoles) in water (30 ml) was refluxed for 16 hours. The brown suspensionwas cooled and filtered over celite. The residue was washed with 20 mlof aqueous 1 N NaOH, and the pale yellow filtrate was acidified to pH 1with aqueous 37% HCl while stirring vigorously. The precipitate wasfiltered, washed with 10 ml of water, and dried under vacuum to yield0.76 grams (66%) of Compound 30 as a white powder. The structure andpurity of Compound 30 was confirmed by its ¹H NMR spectrum.

¹H NMR (300 MHz; DMSO): δ=7.16 ppm (doublet, 4H, 3J, 8.0 Hz), 7.61 ppm(doublet, 4H, J, 8.0 Hz) and 8.00 ppm (singlet, 4H).

Preparation of Para-Bromo Diphenyl Glycoluril (FIG. 22, Compound 33)

Six (6) ml of TFA were added to a solution of urea (3.0 grams, 0.05moles) and Compound 32 (7.3 grams, 0.025 moles) in 200 ml of benzene,and the mixture was refluxed for 12 hours using a Dean-Stark trap, untilwater distillation was no longer observed. The resulting white solidproduct was filtered and washed with cold ethanol, and was thereafterdried under high vacuum to yield 8.73 grams (86%) of the product. Thestructure and purity of Compound 33 were confirmed by its ¹H NMRspectrum.

¹H NMR (300 MHz; DMSO): δ=6.95 ppm (doublet, 2H) 7.01-7.11 ppm(multiplet, 5H, aromatic), 7.22 ppm (doublet, 2H) and 7.79 ppm (singlet,4H, NH).

The substituted glycolurils described above are used to prepare acucurbituril trimer, as is described hereinunder.

Preparation of a Cucurbituril Trimer (FIG. 23, Compound 38)

As is exemplified in FIG. 23, an exemplary pathway for forming acucurbituril trimer involves a disubstituted cucurbituril derived frombenzil. Such a pathway generally includes preparation of abenzil-derived glycoluril, as described hereinabove, a formation of aderivatized CB monomer, and a trimerization step.

As a starting material, a benzil-derived glycoluril (FIG. 23, Compound35) is prepared by reacting a para-substituted benzil with urea in a 1:2ratio in benzene and trifluoroacetic acid, according to the exemplarysyntheses are presented hereinabove. In one non-limiting example, one ofthe para-substituents on the benzil is a carboxyl group and the secondis hydrogen (FIG. 23, Compound 34).

Compound 35, is reacted with glycoluril, Compound 2, in a 1:5 ratio, andwith formaldehyde, in the presence of concentrated sulfuric acid, tothereby form the derivatized cucurbituril biphenyl-CB[6] (FIG. 23,Compound 36). Compound 36 is reacted with 1,3,5-benzenetriamine (BTA) ina 3:1 ratio so as to form three amide bonds between each of the carboxylgroups on the derivatized CB[6] and an amine on the BTA, and thus form acucurbituril trimer having a BTA as the assembling unit (FIG. 23,Compound 38).

Preparation of a CB-Protein Conjugate (FIG. 23, Compound 37)

A derivatized cucurbituril, as described above, can alternatively, or inaddition to the above, be attached to a functional moiety such as aprotein. Thus, for example, a biphenyl-derivatized (FIG. 23, Compound36) is reacted with a protein so as to form an amide bond between thecarboxyl functional group on the CB and a lysine side-chain on theprotein. Such a cucurbituril having a functional moiety such as aprotein attached thereto (FIG. 23, Compound 37) can be used in a varietyof applications including, for example, application that requirelabeling of a protein, identification, isolation, purification and/orimmobilization of a protein and so forth, as is detailed hereinabove.

Example 8 Preparation of a Linear Cucurbituril Trimer

In another exemplary pathway for preparing a linear cucurbituril trimer,disubstituted cucurbiturils are prepared and are thereafter attached toan assembling unit, as is presented in FIG. 24.

As an exemplary starting material, a “benzil trimer” (FIG. 24, Compound39) is reacted with urea in a 1:6 ratio, in benzene and trifluoroaceticacid, so as to afford a benzil-derived structure having three glycolurilunits attached thereto. The latter is further substituted at each endwith a carboxyl group, which can be used for further attachment of avariety of functional moieties or for forming cucurbituril assemblies.

Thus, the benzil-derived glycoluril structure is reacted withglycoluril, Compound 2, in a 1:15 ratio, and with formaldehyde, in thepresence of concentrated sulfuric acid, to thereby form a linear,derivatized, cucurbituril trimer (FIG. 24, Compound 40)

Example 9 Preparation of a Cucurbituril Trimer Having a Rigid AssemblingUnit

An exemplary pathway for preparing a cucurbituril trimer having a rigidassembling unit, according to the present invention, is presented inFIG. 25.

Coronene-1,2,5,6,9,10-hexaone (FIG. 25, Compound 41), a derivative ofcoronene, is reacted with urea, so as to afford an assembling unit whichincludes three glycolurils units fused therein.

The assembling coronene unit is then reacted with glycoluril, Compound2, in a 1:15 ratio, and with formaldehyde, in the presence ofconcentrated sulfuric acid, to thereby obtain the rigid derivatizedcucurbituril trimer (FIG. 25, Compound 42).

Example 10 Synthesis of Rigid Polyacetylene-ContainingPolyamine-Structures and Affinity Pairs Thereof

As is discussed hereinabove, polyamine structures according to thepresent invention, which are characterized by a rigid structure, arehighly advantageous in terms of enhancing the affinity binding tocucurbiturils.

Such rigid polyamine structures have been obtained by designing andpreparing diamine structures that contain a rigid threading moiety. Therigidity of the threading moiety has been obtained by the incorporationof a polyacetylene chain therein. As is described below, these rigidpolyamine structures were found to form stable affinity pairs withCB[6]s.

Following is a detailed description of the various synthetic stages inthe preparation of exemplary rigid polyamine structures according to thepresent invention and of CB-PA affinity pairs containing same. Figures

Preparation of 1-Iodoprop-1-yn-3-ol (FIG. 26, Compound 43)

Compound 43 was prepared according to Cowell and Stille, J. Am. Chem.Soc., 1980, 102, 4193-4198. In brief, a 1.6 M solution of n-butyllithium(117.5 ml, 187.5 mmoles) in hexane was slowly added to a stirredsolution of propargyl alcohol (5 grams, 89.3 mmoles) in 300 ml of THF at−78° C. After the addition was completed, the resulting solution wasfurther stirred for additional 30 minutes. Subsequently, a solution ofiodine (24.95 grams, 98.2 mmoles) in 150 ml of THF was slowly added tothe stirred solution. The mixture was allowed to warm to roomtemperature and was then poured onto a mixture of ice and dilutehydrochloric acid. After separation of the organic layer from theaqueous layer, the THF was removed under vacuum, and the residue wasextracted with three portions of 300 ml diethyl ether. The ether layerwas washed with aqueous solutions of sodium bisulfite and sodiumbicarbonate and with water and was then dried over sodium sulfate. Theether was thereafter removed under vacuum, yielding a crude product,which was purified by flash column chromatography, using a solution of20% ethyl acetate in hexane as eluent, so as to afford Compound 43 as alow melting point pale yellow colored solid (14.8 grams, 91%). Thestructure and purity of Compound 43 were confirmed by its NMR spectra.

¹H NMR (CDCl₃): δ=4.42 (s, 1H), 1.79 (s, 1H) ppm;

¹³C NMR (CDCl₃): δ=92.4, 52.4, 2.8 ppm.

Preparation of Hexa-2,4-diyn-1,6-diol (FIG. 26, Compound 44)

Copper iodide (104 mg, 0.5 mmoles) was added to a stirred solution ofiodo-propargyl alcohol (1 gram, 5.5 mmoles) and propargyl alcohol (0.62grams, 11 moles) in 5 ml of pyrrolidone, at 0° C. under argonatmosphere. The resulting mixture was stirred at room temperature for 30minutes, and was thereafter quenched with an aqueous solution ofammonium chloride and extracted with diethyl ether. The organic extractwas dried over sodium sulfate and the solvent was removed, so as toafford the crude product, which was purified by flash columnchromatography using a solution of 40% ethyl acetate in hexane aseluent, to afford Compound 44 as a colorless semi solid (0.53 grams,88%). The structure and purity of Compound 44 were confirmed by its NMRspectra.

¹H NMR (DMSO): δ=5.39 (t, J=4.5 Hz, 2H), 4.16 (d, J=4.5 Hz, 4H) ppm;

¹³C NMR (DMSO): δ=79.6, 67.9, 49.3 ppm.

Preparation of Hexa-2,4-diyn-1,6-diammonium dichloride (FIG. 26,Compound 45)

Compound 45 was prepared according to Fabiano et al., Synthesis, 1987,190-192. In brief, to a solution of diacetylene diol (DIEA, 0.16 gram,1.45 mmol) in THF (3 ml), a 1 M solution of hydrazoic acid was added. Ina three-necked round bottomed flask equipped with a thermometer and aguard tube, a paste was prepared from NaN₃ (3.25 grams, 50 mmol) andwarm water (3.25 ml), benzene (20 ml) was added thereto and theresulting mixture was cooled to 0° C. Concentrated H₂SO₄ (1.4 ml, 0.5equivalents to NaN₃) was then added dropwise, while maintaining thetemperature below 10° C. Upon completing the addition, the mixture wascooled to 0° C., the organic layer was decanted and dried over sodiumsulfate. Benzene (1.75 ml) was then added, followed by a solution ofDIEA (0.323 gram, 1.6 mmol) in THF (1 ml) and a solution of TPP (0.842gram, 3.2 mmol) in THF (2 m), while stirring. The reaction isexothermic. After stirring the reaction mixture for 1 hour at roomtemperature, and for 3 hours at 50° C., water (0.5 ml) was added, whilemaintaining the temperature at 50° C. for additional 3 hours. Thesolvent was thereafter removed under reduced pressure and the residuewas partitioned between dichloromethane (15 ml) and 1 N hydrochloricacid (15 ml). The aqueous phase was extracted with dichloromethane (3×15ml), and the water was removed under reduced pressure to give the crudeamine hydrochloride. Re-precipitation with a mixture of methanol andether solvent afforded the diamine salt Compound 46 as a brown coloredsolid (0.19 gram, 73% yield).

¹H NMR (DMSO): δ=8.74 (br s, 6H), 3.9 (d, J=6 Hz, 4H) ppm;

¹³C NMR (DMSO): δ=73.4, 69.1, 28.6 ppm.

Preparation of a Hexa-2,4-diyn-1,6-diammonium dichloride-CB[6] Complex(FIG. 26, Compound 46)

To a solution of Compound 46 in water, unsubstituted CB[6] was added andthe reaction mixture was stirred overnight at room temperature. Themixture was then filtered and the filtrate was concentrated to give thecrude product, which was recrystallized by dissolving in a minimalamount of water, followed by precipitation in methanol, filtration,washing with methanol and drying, so as to afford the pure inclusioncomplex.

¹H NMR (D₂O): δ=5.74 (m, 12H), 5.57 & 5.55 (2s, 12H), 4.28 (m, 12H),3.33 (s, 4H) ppm.

X-Ray crystallography of the resulting complex, presented in FIGS. 32a-b, demonstrated a symmetrical host-guest complex, in which threeconformations coexist in a single crystal.

Preparation of Hex-3-yne-1,6-diol (FIG. 27, Compound 47)

Compound 47 was prepared according to Delorme et al. J. Org. Chem. 1989,54, 3635-40. In brief, a 1.6 M solution of n-BuLi (26.8 ml) was added toa solution of butynol (1.1 ml, 14.3 mmoles) in THF (100 ml) at −78° C.,and the reaction mixture was stirred for 1 hour. Neat ethylene oxide(1.5 ml, 28.6 mmoles) was then added at −78° C. and the resultingmixture was allowed to reach room temperature and was then stirred atroom temperature for 30 minutes. The reaction mixture was thereafterquenched with aqueous NH₄Cl (100 m) and ether (200 ml) was addedthereto. The ether layer was separated and the aqueous layer wasextracted with ether (2×50 ml). The combined ether layer was washed withwater and brine, dried and concentrated to give a crude product, whichwas purified by flash column chromatography using a solution of 50%ethyl acetate in hexane as eluent, so as to afford the pure product (1.2gram, 74% yield).

¹H NMR (DMSO): δ=3.68 (t, J=6.7 Hz, 2H), 3.56 (br s, 1H), 2.4 (t, J=6.7Hz, 2H) ppm;

¹³C NMR (DMSO): δ=79.1, 61.2, 22.8 ppm.

Preparation of Hex-3-yne-1,6-diammonium dichloride (FIG. 27, Compound48)

Following the same procedure described above for Compound 45, Compound48 was synthesized using Compound 47 as a starting material (79% yield).

¹H NMR (D₂O): δ=3.12 (t, J=7.5 Hz, 4H), 2.59 (t, J=7.5 Hz, 4H) ppm.

Preparation of a Hex-3-yne-1,6-diammonium dichloride-CB[6] Complex

Following the same procedure described above for compound 46, thisinclusion complex was prepared using Compound 48 and unsubstituted CB[6]as the starting material.

¹H NMR (D₂O): δ=5.80 (d, J=16 Hz, 12H), 5.63 (s, 12H), 4.37 (d, J=16 Hz,12H), 2.73 (t, J=6.7 Hz, 4H), 2.21 (t, J=7 Hz, 4H) ppm.

Preparation of Propargylamido Propanoate (FIG. 28, Compound 49)

To a solution of propargyl amine (5 grams, 0.09 mol) in dichloromethane(DCM, 50 ml), triethyl amine (19 ml, 0.14 mol) was added. The resultingmixture was cooled to 0° C. and a solution of propionyl chloride (9.4ml, 0.109 mol) in DCM (50 ml) was added dropwise thereto. Uponcompletion of the addition, the reaction mixture was stirred at roomtemperature for 2 hours and was thereafter quenched with water (20 ml).The organic layer was separated and the aqueous layer was extracted withDCM (3×50 ml). The combined DCM layer was washed with water and brine,dried over sodium sulfate and concentrated to give a crude product,which was purified by flash column chromatography using 20% ethylacetate in hexane as eluent, to afford 7.4 grams (73% yield of an pureproduct).

¹H NMR (CDCl₃): δ=6.27 (br s, 1H), 4.01 (dd, J=4.5, 3 Hz, 2H), 2.23 (q,J=9.5 Hz, 2H), 2.21 (t, J=6.5 Hz, 1H), 1.13 (t, J=9.5 Hz, 3H) ppm;

¹³C NMR (CDCl₃): δ=173.6, 79.7, 71.2, 29.2, 28.9, 9.5 ppm.

Preparation of Compound 50 (FIG. 28)

Following the same procedure described above for Compound 44, Compound50 was obtained in a 58% yield, using Compound 43 and Compound 49 as thestarting materials.

¹H NMR (DMSO): δ=8.29 (br t, J=4.8 Hz, 1H), 5.41 (t, J=5.4 Hz, 1H), 4.15(d, J=5.7 Hz, 2H), 3.97 (d, J=5.4 Hz, 2H), 2.1 (q, J=7.5 Hz, 2H), 0.98(t, J=7.5 Hz, 3H) ppm.

¹³C NMR (DMSO): δ=172.8, 78.6, 77.3, 67.9, 65.6, 49.3, 28.4, 28.2, 9.7ppm.

Preparation of Compound 51 (FIG. 28)

To a solution of Compound 50 (0.4 gram, 2.4 mmol) in DCM (5 ml),triethyl amine (0.5 ml, 3.6 mmol) and mesyl chloride (0.23 ml, 2.9 mmol)were added, while maintaining the mixture temperature at 0° C. Thereaction mixture was then stirred at room temperature for 1.5 hours andwas thereafter quenched with water (10 ml). The organic layer wasseparated and the aqueous layer was extracted with DCM (2×20 ml). Thecombined organic layer was washed with aqueous NaHCO₃ solution, waterand brine, dried over sodium sulfate and concentrated to give the crudeproduct, which was purified by flash column chromatography using 50%ethyl acetate in hexane as eluent, to afford 0.51 gram (86% yield) ofthe pure product.

¹H NMR (CDCl₃): δ=6.25 (br s, 1H), 4.97 (s, 2H), 4.21 (m, 2H), 3.19 (s,3H), 2.30 (q, J=6.5 Hz, 2H), 1.21 (t, J=6.5 Hz, 3H) ppm;

¹³C NMR (CDCl₃): δ=174.0, 78.7, 73.9, 69.8, 66.6, 58.1, 39.4, 29.9,29.7, 9.9 ppm.

Preparation of Compound 52 (FIG. 28)

Compound 52 was prepared according to Dallanoce et al., Bioorg. & Med.Chem., 1999, 7, 1539-1547. In brief, to a stirred solution of Compound51 (0.2 gram, 0.82 mmol) in DMF (2 ml), pyrrolidone (0.14 ml, 1.65 mmol)was added and the resulting mixture was stirred at room temperature,while monitoring the reaction by TLC. once the reaction was completed,the mixture was quenched with water (8 ml) and extracted with ether(3×30 ml). The ether layer was washed with brine, dried over sodiumsulfate and concentrated, to give the crude product, which was purifiedby flash column chromatography using 40% ethyl acetate in hexane as aneluent, to afford 0.098 gram (55% yield of pure product).

¹H NMR (CDCl₃): δ=6.70 (br s, 1H), 4.03 (d, J=4.5 Hz, 2H), 3.42 (s, 2H),2.54 (m, 4H), 2.16 (q, J=6.5 Hz, 2H), 1.73 (m, 4H), 1.08 (t, J=6.5 Hz,3H) ppm.

¹³C NMR (CDCl₃): δ=173.6, 74.7, 73.5, 68.5, 67.2, 52.2, 43.2, 29.4,29.0, 23.6, 9.5 ppm.

Preparation of Compound 53 (FIG. 28)

Compound 53 was prepared according to Soroka, M., Synthesis, 1989,547-548. In brief, Compound 52 (0.098 gram, 0.45 mmol) was treated withan aqueous solution of 8 M HCl (4 ml) and the solution was refluxedovernight. The reaction mixture was allowed to cool to room temperatureand was then extracted with DCM (2×10 ml). The aqueous layer wasseparated and the water was evaporated. The residue was dissolved inMeOH and was precipitated out with ether. The precipitate was filteredand dried to give the diamine-hydrochloride salt product (0.80 gram, 76%yield).

¹H NMR (D₂O): δ=4.23 (s, 2H), 3.96 (s, 2H), 3.67 (m, 2H), 3.22 (m, 2H),2.15 (m, 2H), 2.01 (m, 2H) ppm;

¹³C NMR (D₂O): δ=73.3, 73.1, 71.2, 70.4, 55.4, 45.3, 31.1, 24.6 ppm.

Preparation of a Compound 53-CB[6] complex (Affinity Pair)

Following the same procedure described above for Compound 46, thisinclusion complex was prepared using Compound 53 and unsubstituted CB[6]as the starting materials.

¹H NMR (D₂O): δ=7.37 (br s, 1H), 7.24 (br s, 3H), 5.57 (2d, J=13.5 Hz,12H), 5.48 (s, 12H), 4.34 (2d, J=13 Hz, 12H), 4.34 (m, 2H), 4.18 (m,2H), 3.83 (m, 2H), 3.09 (m, 2H), 2.02 (m, 4H) ppm.

Preparation of Compound 54 (FIG. 28)

Compound 54 was obtained in a 82% yield as described above for Compound52, using Compound 51 and amine as the starting material.

¹H NMR (CDCl₃): δ=7.11 (m, 3H), 7.02 (m, 1H), 6.19 (br s, 1H), 4.10 (d,J=5 Hz, 2H), 3.75 (s, 2H), 3.58 (s, 2H), 2.92 (t, J=4.5 Hz, 2H), 2.82(t, J=4.5 Hz, 2H), 2.21 (q, J=6.5 Hz, 2H) ppm;

¹³C NMR (CDCl₃): δ=173.4, 134, 133.4, 128.5, 126.4, 126.1, 125.6, 74.1,73.9, 69.6, 67.4, 54.2, 49.6, 47.2, 29.5, 29.2, 29.0, 9.5 ppm.

Preparation of Compound 55 (FIG. 28)

Compound 55 was obtained in 75% yield, using the procedure describedabove for Compound 53, using Compound 54 as the starting material.

¹H NMR (D₂O): δ=7.36 (m, 3H), 7.21 (m, 1H), 4.56 (m, 2H), 4.37 (s, 2H),3.99 (s, 2H), 3.34 (s, 2H), 3.24 (m, 2H) ppm;

¹³C NMR (D₂O): δ=131.2, 129.6, 129.3, 128.0, 127.59, 127.55, 73.9, 72.9,70.4, 68.6, 53.5, 50.6, 46.4, 30.4, 25.7 ppm.

Preparation of Compound 55-CB[6] Complex (Affinity Pair)

Following the same procedure described above for Compound 46, thisinclusion complex was prepared using Compound 55 and unsubstituted CB[6]as the starting materials.

¹H NMR (DMSO): δ=7.24 (m, 4H), 5.55 (d, J=15 Hz, 12H), 5.47 (s, 12H),4.33 & 4.31 (2d, J=18.5, 18.5 Hz, 12H), 4.33 (m, 2H), 4.26 (m, 1H), 4.22(m, 2H), 3.85 (m, 2H), 3.57 (m, 2H), 3.17 (d, J=5 Hz, 2H) ppm.

Preparation of Compound 56 (FIG. 29)

Compound 56 was prepared according to López et al., Org. Lett., 2003, 5,3725-3728. In brief, to a mixture of trimethylsilylacetylene (1.2 ml,8.2 mmol) and Compound 43 (1 gram, 5.5 mmol) in piperidine (10 ml), at0° C., CuCl (27 mg, 0.27 mmol) was added and the reaction mixture wasstirred at room temperature for 30 minutes. The reaction was thenquenched with an aqueous solution of NH₄Cl (20 ml) and the organic layerwas extracted with ether (3×50 ml). The ether layer was dried oversodium sulfate and concentrated to give the crude product, which waspurified by flash column chromatography using 20% ethyl acetate inhexane as eluent, affording 0.46 gram (55%) of the pure product.

¹H NMR (CDCl₃): δ=4.33 (s, 2H), 1.92 (br s, 1H), 0.21 (s, 9H). ¹³C NMR(CDCl₃): δ 87.7, 87.1, 75.8, 70.6, 51.4, −0.5 ppm.

Preparation of Compound 57 (FIG. 29)

Compound 57 was prepared according to López et al., Org. Lett., 2003, 5,3725-3728. In brief, to a solution of Compound 56 (0.5 gram, 3.3 mmol)in a 1:1 mixture of MeOH/THF (4 ml) K₂CO₃ (1.84 grain, 13.1 mmol) wasadded and the reaction was stirred for 30 minutes. The mixture wasdiluted with a solution of 1:2 EtOH/H₂O (12 ml) and was thereafterextracted with ether (4×30 ml). The ether layer was washed with H₂O andbrine, dried (Na₂SO₄) and concentrated, to give the product, which wasused for the next step without purification.

Preparation of Compound 58 (FIG. 29)

Following the procedure described above for Compound 42, Compound 58 wasobtained in 88% yield using Compound 43 and Compound 57 as the startingmaterials.

¹H NMR (DMSO): δ=5.75 (s, 2H), 4.17 (s, 4H). ¹³C NMR (DMSO): δ 79.6,67.9, 54.9, 49.3 ppm.

Preparation of Compound 60 (FIG. 30)

To a dichloromethane solution (3 ml) of Compound 50 (0.3 gram, 1.82mmol), MnO₂ (1.6 gram, 18.6 mmol) was added and the reaction mixture wasstirred at room temperature overnight. The reaction mixture was thenfiltered and the filtrate was concentrated to give the crude product,which was purified by flash column chromatography using 20% ethylacetate in hexane as eluent to afford 0.21 gram (70%) of the pureproduct.

¹H NMR (CDCl₃): δ=9.16 (s, 1H), 6.41 (br s, 1H), 4.21 (d, J=5 Hz, 2H),2.24 (q, J=6 Hz, 2H), 1.15 (t, J=6 Hz, 3H) ppm;

¹³C NMR (CDCl₃): δ=175.8, 173.7, 86.5, 78.8, 73.8, 65.5, 53.4, 29.7, 9.5ppm.

Preparation of Compound 61 (FIG. 30)

Compound 60 was reacted with a mono BOC-protected amine (0.44 gram, 1.18mmol) in DCM (2 ml), after neutralizing the p-tosic acid salt of themono protected amine with triethylamine (0.27 ml, 1.96 mmol). Thereaction mixture was stirred at room temperature overnight. Thevolatiles were then removed under reduced and the residue was dissolvedin methanol (3 ml). sodium borohydride (0.056 gram, 1.47 mmol) was addedand the resulting mixture was stirred at room temperature for 2 hoursand then quenched with saturated ammonium chloride solution (8 m) andextracted with ether (3×20 ml). The organic layer was washed with waterand brine, dried over sodium sulfate, concentrated and purified by flashcolumn chromatography using 40% ethylacetate in hexane as eluent, toafford the pure product (0.15 gram, 44% yield).

¹H NMR (CDCl₃): δ 6.90 (br t, 1H), 4.90 (br t, 1H), 3.99 (d, J=6.5 Hz,2H), 3.37 (s, 2H), 2.98 (m, 2H), 2.56 (t, J=8.5 Hz, 2H), 2.14 (q, J=9.5Hz, 2H), 1.39 (m, 4H), 1.32 (s, 9H), 1.25 (m, 2H), 1.04 (t, J=9.5 Hz,3H) ppm;

¹³C NMR (CDCl₃): δ=173.6, 155.9, 78.7, 77.3, 67.4, 66.9, 53.3, 8.2,40.2, 38.4, 29.6, 29.3, 29.1, 28.9, 28.1, 24.2, 9.5 ppm.

Preparation of Compound 62 (FIG. 30)

A mixture of Compound 61 (0.15 gram, 0.43 mmol) in 6N HCl (5 ml) wasrefluxed overnight. The reaction mixture was then cooled to roomtemperature, DCM was added and the aqueous layer was separated andconcentrated, to give the crude product, which was recrystallized bydissolution in methanol and precipitation by ether to afford 0.095 gramsof the pure product, (73% yield).

¹H NMR (D₂O): δ=4.07 (s, 2H), 3.98 (s, 2H), 3.17 (t, J=7.5 Hz, 2H), 3.01(t, J=7.5 Hz, 2H), 1.72 (m, 4H), 1.46 (m, 2H) ppm.

Preparation of a Compound 62-CB[6] Complex (Affinity Pair)

Following the procedure described above for Compound 46, this inclusioncomplex was prepared using Compound 62 and unsubstituted CB[6] as thestarting materials.

¹H NMR (D₂O): δ=5.745 & 5.741 (2d, J=15.5, 15.5 Hz, 12H), 5.62 (s, 12H),4.37 & 4.35 (2d, J=15.5, 15.5 Hz, 12H), 4.25 (s, 2H), 3.99 (s, 2H), 2.64(m, 4H), 0.69 (m, 4H), 0.39 (m, 2H) ppm.

Preparation of Compound 63 (FIG. 31)

To a solution of propargyl alcohol (2 grams, 35.7 mmol) in DCM (20 ml)Et₃N (7.5 ml, 53.7 mmol) was added and the reaction mixture was cooledto 0° C. Mesyl chloride (3.3 ml, 42 mmol) was then added and theresulting mixture was stirred at room temperature for 1 hour. Thereaction mixture was quenched with aqueous sodium bicarbonate solutionand was then extracted with DCM. The organic layer was washed with waterand brine, dried over sodium sulfate and purified by flash columnchromatography using 30% ethyl acetate in hexane as eluent, to afford4.2 grams (88% yield) of the pure product.

¹H NMR (CDCl₃): δ=4.83 (d, J=2.5 Hz, 2H), 3.11 (s, 3H), 2.71 (t, J=2.5Hz, 1H). ¹³C NMR (CDCl₃): δ 77.8, 75.7, 57.2, 38.9 ppm.

Preparation of Compound 64 (FIG. 31)

To a solution of Compound 63 (0.5 gram, 3.75 mmol) in DMF (4 ml), amine(0.95 ml, 7.52 mmol) was added and the reaction mixture was stirred atroom temperature overnight. The reaction was quenched with water and themixture was extracted with ether. The ether layer was washed with waterand brine, dried over sodium sulfate and purified by flash columnchromatography using 40% ethylacetate and hexane as eluent to afford0.63 gram (98% yield) of the pure product.

¹H NMR (CDCl₃): δ=7.13 (m, 3H), 7.04 (m, 1H), 3.77 (s, 2H), 3.52 (d,J=1.5 Hz, 2H), 2.95 (t, J=5 Hz, 2H), 2.84 (t, J=5 Hz, 2H), 2.28 (br t,1H) ppm.

¹³C NMR (CDCl₃): δ=134.4, 133.6, 128.6, 126.5, 126.1, 125.6, 78.6, 73.2,54.2, 49.6, 46.7, 29.2 ppm.

Preparation of Compound 65 (FIG. 31)

Following the procedure described above for Compound 44, Compound 65 wasobtained in a 62% yield, using Compound 43 and Compound 64 as thestarting materials.

¹H NMR (CDCl₃): δ=7.13 (m, 3H), 7.04 (m, 1H), 4.29 (s, 2H), 3.81 (s,2H), 3.64 (s, 2H), 2.97 (t, J=7 Hz, 2H), 2.88 (t, J=7 Hz, 2H), 2.47 (brs, 1H) ppm.

¹³C NMR (CDCl₃): δ=133.7, 133.3, 128.6, 126.6, 126.3, 125.8, 76.1, 74.9,69.83, 69.78, 54.1, 51.2, 49.6, 47.3, 28.9 ppm.

Preparation of Compound 66 (FIG. 31)

Following the procedure described above for Compound 60, Compound 66 wasobtained in 68% yield, using Compound 65 as the starting material.

¹H NMR (CDCl₃): δ=9.20 (s, 1H), 7.13 (m, 3H), 7.03 (m, 1H), 3.78 (s,2H), 3.71 (s, 2H), 2.95 (t, J=5 Hz, 2H), 2.86 (t, J=5 Hz, 2H).

¹³C NMR (CDCl₃): δ=175.8, 133.9, 133.4, 128.7, 126.5, 126.4, 125.8,86.8, 79.1, 73.7, 68.4, 54.3, 49.8, 47.6, 29.1 ppm.

Preparation of Compound 67 (FIG. 31)

Following the procedure described above for Compound 61, Compound 67 wasobtained in 42% yield, using Compound 66 as the starting material.

¹H NMR (CDCl₃): δ=7.11 (m, 3H), 7.02 (m, 1H), 4.60 (br s, 1H), 3.76 (s,2H), 3.58 (s, 2H), 3.48 (br s, 2H), 3.09 (m, 2H), 2.92 (t, J=4.5 Hz,2H), 2.83 (t, J=4.5 Hz, 2H), 2.67 (m, 2H), 1.47 (m, 4H), 1.43 (s, 9H),1.35 (m, 2H) ppm.

¹³C NMR (CDCl₃): δ=155.9, 134.2, 133.5, 128.6, 126.5, 126.1, 125.6,78.9, 73.6, 69.8, 67.8, 54.2, 49.6, 48.4, 47.3, 40.4, 38.6, 29.9, 29.6,29.3, 29.1, 28.4, 24.4 ppm.

Preparation of Compound 68 (FIG. 31)

Following the procedure described above for Compound 62, Compound 68 wasobtained in 75% yield, using Compound 67 as the starting material.

¹H NMR (D₂O): δ=7.33 (m, 3H), 7.21 (m, 1H), 4.65 (m, 2H), 4.47 (m, 1H),4.38 (s, 2H), 4.09 (s, 2H), 3.86 (m, 1H), 3.25 (m, 2H), 3.15 (t, J=6.5Hz, 2H), 2.98 (t, J=6 Hz, 2H), 1.71 (m, 4H), 1.46 (m, 2H) ppm.

¹³C NMR (D₂O): δ 130.6, 129.01, 128.7, 127.4, 127.0, 126.9, 73.1, 71.1,70.6, 68.4, 52.9, 50.1, 46.9, 45.8, 39.3, 37.0, 26.4, 25.1, 23.9, 22.9ppm.

Preparation of a Compound 68-CB[6] Complex (Affinity Pair)

Following the procedure described above for Compound 46, this inclusioncomplex was prepared using Compound 68 and unsubstituted CB[6] as thestarting materials.

¹H NMR (D₂O): δ=7.34 (m, 3H), 7.19 (m, 1H), 5.73 & 5.71 (2d, J=13.5 Hz,12.5 Hz, 12H), 5.58 (s, 12H), 4.59 (s, 2H), 4.34 (m, 15H), 4.24 (m, 1H),3.86 (m, 1H), 3.62 (m, 1H), 3.23 (m, 2H), 2.72 (t, J=7 Hz, 2H), 2.56 (t,J=7.5 Hz, 2H), 0.67 (m, 4H), 0.36 (m, 2H) ppm.

Example 11 An Affinity Labeling Using a Polyamine-Cucurbituril AffinityPair

An affinity labeling procedure involves attaching a labeling moiety to aspecific target biomolecule, which is present in a heterologous mixture,such that only the desired target biomolecule will be labeled with thelabeling moiety. The affinity labeling procedure according to thepresent invention allows either the polyamine structure or thecucurbituril assembly to be attached to the target biomolecules or thelabeling moiety.

An exemplary procedure according to the present invention, involvesattaching a polyamine structure to the target biomolecule, via, forexample, a reactive amine or amide group thereof. For example, in casewhere the biomolecule is a protein, as is exemplified in FIG. 7, thepolyamine structure can be attached to an amine group of a lysineside-chain or to the amide group at the N-terminus. The polyaminestructure can be, for example, comprised of two threading moietiesterminating with amino groups, which are capable of binding specificallyto an assembly of two cucurbiturils, as is illustrated in FIG. 7.Respectively, a cucurbituril assembly of two derivatized CB unitsattached to an assembling unit, is attached, via the assembling unit, toa labeling moiety, e.g., a fluorescent compound, as is illustrated inFIG. 7.

The polyamine structure and the cucurbituril assembly are designed so asto have specific chemical and spatial matching therebetween, such thateach threading moiety of the polyamine structure undergoes complexationwith each of the CB units in the cucurbituril assembly, and the doublecomplexation event exhibits a dissociation constant of about 10⁻¹² M orless.

Thus, by contacting the polyamine structure that is attached to e.g., aprotein, with a cucurbituril assembly that is attached to e.g., afluorescent compound, the protein molecules are labeled by thefluorescent compound via an affinity pair that exhibits a lowdissociation constant and provides a highly efficient affinity labeling.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1. An affinity pair comprising a cucurbituril assembly, and a polyaminestructure being capable of binding thereto, said cucurbituril assemblycomprising at least two cucurbiturils and at least one assembling unit,said assembling unit being covalently attached to each of saidcucurbiturils, and said polyamine structure comprising at least twothreading moieties terminating and/or interrupted by at least two aminogroup, said threading moieties being covalently attached therebetweenvia a branching unit, said polyamine structure being suitably sized tosaid cucurbituril assembly.
 2. The affinity pair of claim 1, whereinsaid cucurbituril assembly further comprises at least one functionalmoiety attached thereto.
 3. The affinity pair of claim 2, wherein saidat least one functional moiety is selected from the group consisting ofa pharmaceutically active agent, a biomolecule, and a labeling moiety.4. The affinity pair of claim 2, wherein said at least one functionalmoiety forms a part of a solid support.
 5. The affinity pair of claim 1,wherein said polyamine structure further comprises at least onefunctional moiety attached thereto.
 6. The affinity pair of claim 5,wherein said at least one functional moiety is selected from the groupconsisting of a pharmaceutically active agent, a biomolecule, and alabeling moiety.
 7. The affinity pair of claim 5, wherein said at leastone functional moiety forms a part of a solid support.
 8. The affinitypair of claim 1, having a dissociation constant lower than 10⁻⁶ M.